Chimeric proteins for use in transport of a selected substance into cells

ABSTRACT

Chimeric proteins, which bind a cell surface receptors, useful in transporting a selected substance present in extracellular fluids, such as blood or lymph, into cells; quantitative assays for the selected substance using chimeric proteins; DNA encoding the chimeric proteins; plasmids which contain DNA encoding the chimeric proteins; mammalian cells, modified to contain DNA encoding the chimeric proteins, which express and, optionally, secrete the chimeric proteins; a method of producing the chimeric proteins; a method of isolating the chimeric proteins; a method of using the chimeric proteins to assay the selected substance; and a method of reducing extracellular levels of the selected substance through administration of the chimeric protein, which results in transport of the selected substance into cells.

DESCRIPTION Background of the Invention

The transport of molecules across cell membranes is an importantcomponent of the physiologic mechanisms that mediate homeostasis at thelevels of the cell and the organism as a whole. Molecules that are useddirectly or indirectly in the assembly of cellular components aretransported from the extracellular fluid into the cell, usually by theaction of specific cell-surface receptors which bind to the selectedsubstance and mediate its uptake into specific cell types. Manyhormones, enzymes, and drugs which influence cellular activity are alsotransported into cells by specific cell-surface receptors. Furthermore,toxic molecules which are either produced in the body (i.e. throughnormal or defective metabolic pathways) or introduced by ingestion orexposure can be taken up and sequestered or metabolized by certaincells.

Removal of substances, both endogenously-produced and foreignsubstances, from extracellular fluids, such as blood or lymph, is oftenphysiologically appropriate. However, in many instances, removal isimpaired or occurs to a lesser extent than desirable and disease occurs.An example concerns LDL cholesterol, a naturally-occurring substancewhich must be removed at a controlled rate if abnormally elevated levelsand the accompanying adverse effects are to be avoided.Hypercholesterolemia in humans is a condition characterized by elevatedlevels of total serum cholesterol. It is usually caused by an excess oflow density lipoprotein (LDL) cholesterol or a deficiency of highdensity lipoprotein (HDL) cholesterol and often leads to atherosclerosisand coronary artery disease. LDL is continually formed in the blood fromapolipoproteins produced by the liver. In order to maintain asteady-state level, LDL is removed from the blood at a rate equal to itsformation. If LDL removal is impaired, the blood level of LDL increasesand atherosclerosis is a greater risk. Atherosclerosis is by far theleading cause of death in the United States, accounting for overone-half of all deaths. (Harrison's Principles of Internal Medicine, Ed.J. D. Wilson et al., 12th ed., p. 995, McGraw-Hill, New York, 1991).

LDL particles carry approximately 60-70% of total serum cholesterol. LDLis a large spherical particle with an oily core composed ofapproximately 1500 cholesterol molecules, each of which is linked to along-chain fatty acid by an ester linkage. Surrounding the core is alayer of phospholipid and unesterified cholesterol molecules, arrangedin such a manner that the hydrophilic heads of the phospholipids are onthe outside, and thus making it possible for the LDL to be dissolved inblood or intercellular fluid. Each LDL particle contains one molecule ofApolipoprotein B-100 (ApoB-100), a large protein molecule which isembedded in the hydrophilic coat of LDL. ApoB-100 is recognized andbound by the LDL receptor, which is present on the surfaces of cells.LDL bound to a LDL receptor is carried into the cell, in which the twoare separated. The LDL receptor is recycled to the cell surface and theLDL is delivered to a lysosome. In the lysosome, LDL is processed toliberate unesterified cholesterol. The liberated cholesterol isincorporated into newly synthesized cellular membranes in all cells and,in specialized cells, is used for other purposes (e.g., steroid hormonesynthesis, bile acid production).

The steady-state level of serum LDL is determined to a large extent bythe number of functional hepatic LDL receptors (LDLRs), which play acentral role in the removal of circulating LDL. (Brown, M. S. andGoldstein, J. L., Science, 232:34-47 (1986)) Individuals with familialhypercholesterolemia (FH) may be either heterozygous or homozygous formutations leading to defective LDLRs and, as a result, these individualshave excess serum LDL. Other individuals who have elevated serum LDLlevels may carry leaky or previously uncharacterized LDLR mutations ormight be producing too much LDL due to elevated intake of dietary fat.Both FH and non-FH patients have elevated cardiovascular risk and couldbenefit from a therapy based on increasing the catabolism of LDL as aresult of increased cellular uptake.

Thus, there exists a need to develop methods for increasing the uptakeof selected substances into cells. These substances may be destined forcatabolism as discussed above, or they may be designed to influenceintracellular processes and thus be considered regulatory agents. Thus,cellular activity may be altered by introducing new regulatory agentswhich can alter specific intracellular processes into recipient cells.For example, cellular patterns of protein phosphorylation, expression ofspecific cellular genes, and cell growth properties may be altered byintroduction of an appropriate regulatory agent into a cell. Theseregulatory agents may be proteins which have enzymatic activity or theymay be proteins that bind specific cellular targets, targets which maybe comprised of nucleic acid, protein, carbohydrate, lipid, orglycolipid.

SUMMARY OF THE INVENTION

Cell surface receptors provide a route for introducing selectedsubstances into cells. The natural ligand of the receptor may be aportion of a chimeric protein in which the ligand domain is functionallylinked to a protein domain that exerts a desired effect within a celland is therapeutic in vivo. Alternatively, the protein domain may bebound to a selected substance which is to be removed from extracellularfluids for catabolism or other metabolic processing.

The present invention relates to chimeric proteins useful intransporting a selected substance present in extracellular fluids, suchas blood or lymph, into cells; quantitative assays for the selectedsubstance using chimeric proteins; DNA encoding the chimeric proteins;plasmids which contain DNA encoding the chimeric proteins; mammaliancells, modified to contain DNA encoding the chimeric proteins, whichexpress and, optionally, secrete the chimeric proteins; a method ofproducing the chimeric proteins; a method of isolating the chimericproteins; a method of using the chimeric proteins to assay the selectedsubstance; and a method of reducing extracellular levels of the selectedsubstance through administration of the chimeric protein, which resultsin transport of the selected substance into cells. The present inventionalso relates to a method of gene therapy, in which mammalian cellsexpressing and secreting the chimeric protein are implanted into anindividual, in whom the chimeric protein is expressed and secreted andbinds the selected substance. The resulting selected substance-chimericprotein complex is taken up into somatic cells and, as a result, theextracellular levels of the selected substance are reduced.

The selected substance can be a normally-occurring (endogenouslyproduced) constituent of the blood, such as a nutrient, metabolite,naturally-occurring hormone or lipoprotein, or a foreign constituent,such as a pathogen, toxin, environmental contaminant or drug orpharmacologic agent. In either case, the selected substance is removedfrom the extracellular fluid, such as blood or lymph, by means of achimeric protein which selectively binds the selected substance and alsobinds a cell surface receptor present on one or more types of somaticcells, particularly human somatic cells. The resulting chimericprotein-selected substance complex binds to the cell surface receptorand is transported into the cell, where it is sequestered ormetabolized, resulting in reduced extracellular levels of the selectedsubstance.

Chimeric proteins of the present invention include at least twocomponents: a functional domain and a carrier domain. The functionaldomain comprises an amino acid (polypeptide) sequence which binds theselected substance to be transported into cells or contains a sequencewhich will affect the target cell in a specific way. The carrier domaincomprises an amino acid (polypeptide) sequence which binds a cellsurface receptor present on one or more types of somatic cells. Theamino acid sequence which is the functional domain can be a ligandbinding domain of the selected substance; the amino acid sequence whichis the carrier domain can bind to a cell-surface receptor and is thus acell surface receptor ligand. Both the functional and carrier domainsmay be modified post-translationally, for example, by glycosylation atcertain sites. In the case in which the selected substance is anormally-occurring constituent of the blood, lymph, or extracellularfluid, the ligand-binding domain which binds the selected substance isan amino acid sequence which normally binds the selected substance(i.e., binds the selected substance in humans), a modified form of sucha sequence with altered binding properties, or an amino acid sequencewhich is not usually found in humans but has been produced by syntheticor genetic engineering methods and binds the selected substance. Forexample, the functional and/or carrier domains may be amino acidsequences selected from a combinatorial peptide library or phage displaylibrary. The functional and/or carrier domains may also comprise theantigen binding domain of an immunoglobulin or single-chain antibody,wherein the antigen binding domain of the immunoglobulin or single-chainantibody recognizes the desired selected substance or cell surfacereceptor. In the case in which the selected substance is a foreignconstituent, the amino acid sequence which binds the selected substanceis one selected from naturally-occurring ligand-binding domains whichbind the foreign constituent or an amino acid sequence designed to bindthe foreign constituent. The amino acid sequence which binds the cellsurface receptor typically binds a cell surface receptor other than thereceptor to which the selected substance normally binds. Thus, themethod causes a selected substance to enter a cell by a route which isdifferent from that which it normally takes in an organism.

The domains of the chimeric protein can be linked in a variety ofconfigurations, as long as the resulting chimeric protein is able tobind both the selected substance and the cell surface receptor.Typically, the two domains are encoded by a single reading frame in arecombinant DNA molecule, and the two domains are linked by a peptidebond. The two domains may be separated by one or more amino acids alsoencoded by the open reading frame. Alternatively, the two domains may beexpressed from separate DNA molecules and become linked in vitro or invivo through either non-covalent (e.g., hydrophobic or ionicinteraction) or covalent (e.g., disulfide) linkage.

Once the selected substance is bound to the ligand binding domain of thechimeric protein, the resulting complex is referred to as the selectedsubstance-chimeric protein complex. The cell surface receptor ligandpresent in the selected substance-chimeric protein complex binds to itscell surface receptor and the complex is transported into the cell(e.g., by endocytosis), thus reducing circulating levels of the selectedsubstance.

In one embodiment, the present invention relates to chimeric proteinsuseful in transporting LDL into cells; pharmaceutical compositionscontaining chimeric proteins; assays for LDL using the chimericproteins; DNA encoding the chimeric proteins; plasmids which contain DNAencoding the chimeric proteins; mammalian cells, modified to contain DNAencoding the chimeric proteins, which express and, optionally, secretethe proteins (genetically modified cells); a method of producing thechimeric proteins; a method of isolating the chimeric proteins; a methodof using the chimeric proteins to assay LDL and a method of reducingextracellular levels of LDL cholesterol (LDL) by administering achimeric protein which transports LDL into cells.

In one embodiment of reducing extracellular LDL levels, a chimericprotein which binds LDL and a cell surface receptor other than LDLreceptor (LDLR) is administered to a human patient in whom serumcholesterol level is to be reduced. In another embodiment of reducingextracellular LDL levels, cells modified to contain DNA which encodesthe chimeric protein, are implanted into an individual. In theindividual, the chimeric protein is expressed in and secreted by thegenetically modified cells, binds LDL in the blood and formsLDL-chimeric protein complexes, which are transported intonon-genetically modified cells, thus reducing serum LDL cholesterollevels.

Chimeric proteins of the present invention useful for LDL transport andfor assaying LDL in a sample include at least two components: afunctional domain, which comprises the amino acid sequence of theligand-binding domain of the LDLR and a carrier domain, which comprisesan amino acid sequence which binds a cell surface receptor other thanthe LDLR on one or more types of somatic cells, particularly humansomatic cells. In one embodiment of the chimeric protein useful forincreasing uptake of LDL into cells, an amino-terminal sequencecomprising the ligand binding domain (i.e., the LDL binding domain) ofthe LDLR is joined to a C-terminal sequence comprising a cell surfacereceptor ligand. The carboxy-terminus of the LDL binding domain isjoined to the amino-terminus of the cell surface receptor ligand domain.

In one embodiment of the chimeric protein, the two components are theligand-binding domain of the human LDLR and human transferrin; the LDLbinding domain of the LDLR is joined to the amino-terminus of the maturehuman transferrin polypeptide. This chimeric protein can bind both LDLand the transferrin receptor. Once bound to the transferrin receptor onthe surface of a cell, such as a liver cell, the chimeric protein andthe LDL bound to the chimeric protein LDLR component are endocytosed bytransferrin receptor-mediated endocytosis, with the result that LDLenters the cell and the extracellular LDL concentration is reduced. Thetransferrin receptor is present on a wide variety of mammalian celltypes, allowing the chimeric protein to promote LDL uptake in a widevariety of mammalian cells.

In a second embodiment, the chimeric protein comprises a functionaldomain which is a ligand-binding domain of LDLR, and a carrier domainwhich is an amino acid sequence which binds a cell surface receptorother than the transferrin receptor, such as the serum albumin receptor,asialoglycoprotein receptor, an adenovirus receptor, a retrovirusreceptor, CD4, lipoprotein (a), immunoglobulin Fc receptor,a-fetoprotein receptor, LDLR-like protein (LRP) receptor, acetylated LDLreceptor, mannose receptor, or mannose-6-phosphate receptor. In general,an amino acid sequence that binds to any receptor which can bind andinternalize bound ligand may be used. These chimeric proteins bind bothLDL and a cell surface receptor and can be used to enhance the uptake ofLDL into a wide variety of cells. Once bound to the receptor, thechimeric protein-LDL complex is endocytosed with the result that the LDLenters the cell and the extracellular LDL concentration is reduced.

A chimeric protein of the present invention is produced by anappropriate mammalian cell which contains DNA encoding the chimericprotein. Modified mammalian cells of the present invention (i.e.,mammalian cells modified to contain nucleic acid encoding a chimericprotein of the present invention) include mammalian cells which arestably or transiently transfected or infected with a plasmid, nucleicacid fragment, or other vector, including a viral vector, comprising DNAor RNA encoding the chimeric protein or which are derived (directly orindirectly) from a progenitor modified mammalian cell which contains DNAor RNA (e.g., plasmid, nucleic acid fragment, or other vector comprisingDNA or RNA) encoding the chimeric protein.

In one embodiment of the present method of producing chimeric proteins,the mammalian cell used is a cell line, such as a Chinese hamster ovary(CHO) cell line, which contains and expresses DNA which encodes thechimeric protein. Optionally, the chimeric protein is secreted into themedium in which the transfected CHO cells are cultured. In a secondembodiment of the present method of producing chimeric proteins, themodified mammalian host cell is a primary or secondary cell, such as aprimary or secondary human fibroblast, transfected or infected with DNAencoding the chimeric protein. The modified cell (e.g., a transfected orinfected primary or secondary human fibroblast) expresses the encodedchimeric protein and, optionally, secretes it into the culture medium.Alternatively, the transfected or infected primary or secondary cellsmay be implanted into an individual, such as a human, in whom thechimeric protein is secreted for therapeutic purposes.

The chimeric protein produced by cultured modified cells may be isolatedfrom cell lysates or the culture medium by any appropriate method. Onesuch method, described herein, is based on affinity chromatography inwhich the chimeric protein is isolated by first binding to an antibodycolumn prepared using an antibody directed against the cell surfacereceptor ligand, eluting, and subsequently by binding to a columnbearing the selected substance to which the ligand binding domain of thechimeric protein binds. For example, the chimeric protein which bindsLDL and the cell surface receptor for transferrin can be isolated byfirst being separated by binding to an anti-transferrin antibody column,and subsequently by binding to LDL bound to an anti-LDL antibody column.As a result of the isolation method, purified intact chimeric protein isobtained. Alternatively, in the first separation step, the chimericprotein is bound to a column bearing the selected substance to which theligand binding domain of the chimeric protein binds and in the secondseparation step, is bound to a column bearing an antibody directedagainst the cell surface receptor ligand. As described herein, chimericprotein which includes domains from both LDLR and transferrin has beenpurified.

The chimeric protein of the present invention is useful to transport theselected substance, LDL, into cells, such as liver cells, thus reducingextracellular levels of the LDL. This has clinical or therapeuticapplications, such as in controlling or lowering serum LDL levels inhumans, such as hypercholesterolemic individuals. In one embodiment ofthe method of the present invention in which LDL is transported intocells through the use of the chimeric protein, cells expressing thechimeric protein are implanted in an individual in whom serum LDL levelsare to be lowered. In the individual, the cells produce the chimericprotein, which enters the interstitial fluid. From the interstitium thechimeric protein can enter the lymphatics and ultimately, thebloodstream, where it binds LDL resulting in formation of a chimericprotein-LDL complex. The complex passes (e.g., via the bloodstream) to acell which bears a transferrin receptor (e.g., a hepatocyte), and isbound to the cell as a result of the transferrin domain-transferrinreceptor interaction. The chimeric protein-LDL complex is taken up bymeans of the transferrin receptor-mediated endocytosis pathway thatnormally functions to internalize transferrin. In another embodiment ofthe method, purified or partially purified chimeric protein isadministered to an individual (particularly a human) in whom increasedLDL transport into cells is desired.

The chimeric protein has a wide variety of other clinical or therapeuticapplications, such as in reducing the circulating levels of normal orabnormal endogenously produced metabolites or nutrients (e.g. acetylatedlow density lipoprotein, apolipoprotein E4, tumor necrosis factor a,transforming growth factor β, a cytokine, an immunoglobulin, a hormone,glucose, a bile salt, a glycolipid such as glucocerebroside whichaccumulates in patients with Gaucher disease or ceramidetrihexosidewhich accumulates in patients with Fabry disease!, or aglycosaminoglycan such as those that accumulate in patients with Hunter,Hurler, or Sly syndromes!) or of foreign substances (e.g., pathogens,environmental contaminants, or alcohol).

The chimeric protein of the present invention is also useful to assay,particularly to quantitatively assay, the selected substance to whichthe ligand binding domain binds. For example, it may be used in an assayto determine levels of LDL, immunoglobulins, growth hormone, andApolipoprotein E in the blood. For example, the chimeric protein can beused as a component in known methods, such as an enzyme-linked assay, toassay a selected substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of LDL uptake via the normal LDLreceptor-mediated pathway (LDLR pathway) and the transferrinreceptor-mediated pathway (TFR pathway).

FIG. 2 is a schematic representation of LDLR/TF expression plasmidpEFBOS/LDLrTF1.S. Specific regions of the plasmid are denoted by theshadings or lines indicated. Restriction endonuclease sites eliminatedas a result of blunting the termini of XbaI-digested pEF-BOS bytreatment with Klenow fragment of E. coli. DNA polymerase and ligationto the EcoNI-SmaI fragment containing the fusion gene in which the EcoNIsite was similarly blunt-ended are in parentheses.

FIGS. 3A-3E are the nucleotide sequence (SEQ ID NO: 15 1) andcorresponding amino acid sequence (SEQ ID NO: 2) for the chimeric cDNAin pEFBOS/LDLrTF1.S, in which the initiating methionine is indicated asthe first amino acid of the fusion protein; . . . indicates thetermination (stop) codon; the underlined codons denote the humantransferrin portion of the chimeric protein; the codons not underlineddenote the LDLR portion of the chimeric protein, nucleotides 1-13: 5'non-coding LDLR sequences; nucleotides 3236-3428:3' non-codingtransferrin sequences.

FIG. 4 is a schematic representation LDLR/TF expression plasmidpEFBOS-LDLR/TF-710, in which specific regions of the plasmid are denotedby the shadings or lines indicated.

FIGS. 5A-5F are the nucleotide (SEQ ID NO: 3) and corresponding aminoacid sequence (SEQ ID NO: 4) for the chimeric cDNA inpEFBOS-LDLR/TF1-710, in which the initiating methionine is indicated asthe first amino acid of the fusion protein; . . . indicates thetermination (stop) codon; the underlined codons denote the humantransferrin portion of the chimeric protein; the codons not underlineddenote the LDLR portion of the chimeric protein; nucleotides 1-13: 5'non-coding LDLR sequences and nucleotides 4244-4603: 3' non-codingtransferrin sequences are shown.

FIG. 6 shows the results of analysis by non-reducing SDS-PAGE andWestern blot analysis of LDLR/TF chimeric protein forms, produced inChinese hamster ovary (CHO) cells, at various stages of immunoaffinity(IA) purification.

FIG. 7 is a graphic representation of specific binding of human LDL toLDLR/TF chimeric protein at the concentrations of LDL indicated.

FIG. 8 is a graphic representation of results of a microplate bindinganalysis of chimeric protein binding to LDL.

FIG. 9 shows the results of Western blot analysis of LDLR/TF chimericproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exemplifies methods for introducing selectedsubstances into cells for in vivo therapy. Further, the inventionteaches the use of gene therapy to produce therapeutic proteins designedto transport selected substances into cells.

As described herein, Applicants have developed a new strategy for uptakeof a selected substance into cells through the use of a chimeric proteinand by means of a mechanism by which the selected substance is notnormally taken up in cells. The chimeric protein binds the selectedsubstance and also binds a cell surface receptor present on somaticcells, particularly on human somatic cells. Binding of the chimericprotein and the selected substance results in formation of a chimericprotein-selected substance complex, which is bound by the cell surfacereceptor and transported into the cell bearing the receptor. Theselected substance can be a normally-occurring (endogenously produced)constituent of extracellular fluid, such as blood or lymph, or a foreignconstituent such as a drug or a toxin. The chimeric protein of thepresent invention is itself delivered or administered to an individualor is provided to an individual by a gene therapy method in which cellswhich express and secrete the chimeric protein are introduced into anindividual, in whom the chimeric protein is produced and secreted. Thechimeric protein selectively binds the selected substance in theextracellular fluid (e.g., blood, lymph) of the individual, thusproducing a selected substance-chimeric protein complex. Theligand-binding domain of the complex is bound by cell surface receptorson somatic cells in the individual. The complex is transported into thecell to which it is bound and the extracellular level of the selectedsubstance is, thus, reduced.

Chimeric Proteins for Reducing Serum LDL Levels

In one embodiment, Applicants have developed a new strategy for uptakeof human LDL which does not normally occur in humans, circumvents thedefective LDLR or supplements the reduced level of LDLR known to occurin individuals with familial hypercholesterolemia, and augments orincreases the ability of cells (those with a normal number of functionalLDLRs and those with abnormal numbers of LDLRs) to take up LDL. This newmethod enhances LDL transport into liver cells, whether they have anabnormal number of functional LDLRs or a normal number of functionalLDLRs. Applicants have determined that a receptor which is present onhuman cell surface membranes can be used as a means by which LDL can betransported into cells, such as liver cells. Cell surface receptors,such as the transferrin receptor, the serum albumin receptor, theasialoglycoprotein receptor, an adenovirus receptor, a retrovirusreceptor, CD4, lipoprotein (a) receptor, immunoglobulin Fc receptor,a-fetoprotein receptor, LDLR-like protein (LRP) receptor, acetylated LDLreceptor, mannose receptor, or mannose-6-phosphate receptor, can be usedas a means by which LDL can be transported into cells. These receptorsare present, respectively, on a wide variety of different cell types andallow uptake of LDL into a wide variety of cell types.

Applicants have produced chimeric proteins useful for transport of LDLinto cells. These chimeric proteins are the subject of the presentinvention, as are nucleic acid sequences encoding the chimeric proteins;mammalian host cells containing DNA (or RNA) encoding the chimericproteins, which is expressed in the cells; a method of producing thechimeric proteins; a method of isolating the chimeric proteins, a methodin which the chimeric proteins are used to quantitatively assay for LDL,and a method of reducing extracellular LDL levels, including atherapeutic method of reducing serum LDL levels in an individual. In thetherapeutic method, a chimeric protein which binds LDL and a human cellsurface receptor other than the human LDLR is provided to an individual,either by administration of the chimeric protein itself or byadministration (e.g., implantation) of cells which express and secretethe chimeric protein. In either case, the chimeric protein binds LDL anda human cell surface receptor and the complex is transported into thecell to which it is bound, reducing extracellular levels of LDL.

In one embodiment, the chimeric protein comprises a first domain, whichis the ligand-binding domain of the LDLR and a second domain, which istransferrin (TF). The human transferrin receptor has a very highaffinity for its ligand; the equilibrium dissociation constant (Kd) is2-7 nM (Trowbridge, I.S. et al., Biochem. Pharmacol., 33:925-993(1984)). Transferrin can be bound to its receptor even when thetransferrin concentration is very low in comparison with total bloodprotein concentration. Similarly, the LDL receptor has a high affinityfor its ligand (Kd=7.2 nM for liver receptors (Krampler, F. et al., J.Clin. Invest., 80:401-408 (1987) and 2.8 nM for fibroblast receptors(Innerarity, T. L. et al., Meth. Enzymol., 129:542-565 (1986)). Thus,the chimeric protein of this embodiment has a high affinity for the twocomponents (human LDL and the human transferrin receptor) which must bebrought together for LDL to be transported into cells by transferrinreceptor-mediated endocytosis. A diagram of how an LDLR-transferrin(LDLR/TF) chimeric protein functions to promote LDL uptake via thetransferrin receptor is shown in FIG. 1. LDL taken up by either pathwayis metabolized to release cholesterol and free amino acids. In theLDLR-pathway, LDLR is recycled to the cell surface after LDL isreleased. In the TFR pathway, LDL is released and the TFR and LDLR/TFchimeric protein are recycled to the cell surface.

The entire human transferrin protein can be present in the chimericprotein; alternatively, only the portions of human transferrin necessaryfor binding to iron and the human transferrin receptor present on humancells are included in the chimeric protein. At neutral pH, each TFRbinds two diferric (i.e., iron-saturated) TF molecules. The binding siteis in the N-terminal domain of each TF. Monoferric TF binds TFR lessreadily, while apoTF (i.e., TF without bound iron) fails to bind TFR. Incontrast, when iron is released in the acidic environment of thelysosome, ApoTF remains bound to TFR in order for recycling of thereceptor and apoTF to the cell surface. As used herein, the term "humantransferrin" refers to the entire human transferrin molecule or thosesegments of the protein necessary for binding to iron and to transferrinreceptors on human cell surface membranes.

In one embodiment, the ligand-binding domain of the human LDL receptoris joined to human transferrin at the N-terminus of the mature TFmolecule. The ligand-binding domain may include other LDL receptorregions which are present in the naturally-occurring receptor protein.

Production of Chimeric Proteins

The chimeric protein of the present invention, such as the chimericprotein for transporting LDL into cells, can be produced using hostcells expressing a single nucleic acid encoding the entire chimericprotein or more than one nucleic acid sequence, each encoding a domainof the chimeric protein and, optionally, an amino acid or amino acidswhich will serve to link the domains. The chimeric proteins can also beproduced by chemical synthesis.

A. Host Cells

Host cells used to produce chimeric proteins are bacterial, yeast,insect, non-mammalian vertebrate, or mammalian cells; the mammaliancells include, but are not limited to, hamster, monkey, chimpanzee, dog,cat, bovine, porcine, mouse, rat, rabbit, sheep and human cells. Thehost cells can be immortalized cells (a cell line) or non-immortalized(primary or secondary) cells and can be any of a wide variety of celltypes, such as, but not limited to, fibroblasts, keratinocytes,epithelial cells (e.g., mammary epithelial cells, intestinal epithelialcells), ovary cells (e.g., Chinese hamster ovary or CHO cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells, hepatocytesand precursors of these somatic cell types.

Cells which contain and express DNA or RNA encoding the chimeric proteinare referred to herein as genetically modified cells. Mammalian cellswhich contain and express DNA or RNA encoding the chimeric protein arereferred to as genetically modified mammalian cells. Introduction of theDNA or RNA into cells is by a known transfection method, such aselectroporation, microinjection, microprojectile bombardment, calciumphosphate precipitation, modified calcium phosphate precipitation,cationic lipid treatment, photoporation, fusion methodologies, receptormediated transfer, or polybrene precipitation. Alternatively, the DNA orRNA can be introduced by infection with a viral vector. Methods ofproducing cells, including mammalian cells, which express DNA or RNAencoding a chimeric protein are described in co-pending patentapplications U.S. Ser. No. 08/334,797, entitled "In Vivo ProteinProduction and Delivery System for Gene Therapy", by Richard F Selden,Douglas A. Treco and Michael W. Heartlein (filed Nov. 4, 1994); U.S.Ser. No. 08/334,455, entitled "In Vivo Production and Delivery ofErythropoietin or Insulinotropin for Gene Therapy", by Richard F Selden,Douglas A. Treco and Michael W. Heartlein (filed Nov. 4, 1994) and U.S.Ser. No. 08/231,439, entitled "Targeted Introduction of DNA Into Primaryor Secondary Cells and Their Use for Gene Therapy", by Douglas A. Treco,Michael W. Heartlein and Richard F Selden (filed Apr. 20, 1994). Theteachings of each of these applications are expressly incorporatedherein by reference.

B. Nucleic Acid Constructs

A nucleic acid construct used to express the chimeric protein can be onewhich is expressed extrachromosomally (episomally) in the transfectedmammalian cell or one which integrates, either randomly or at apre-selected targeted site through homologous recombination, into therecipient cell's genome. A construct which is expressedextrachromosomally comprises, in addition to chimeric protein-encodingsequences, sequences sufficient for expression of the protein in thecells and, optionally, for replication of the construct. It typicallyincludes a promoter, chimeric protein-encoding DNA and a polyadenylationsite. The DNA encoding the chimeric protein is positioned in theconstruct in such a manner that its expression is under the control ofthe promoter. Optionally, the construct may contain additionalcomponents such as one or more of the following: a splice site, anenhancer sequence, a selectable marker gene under the control of anappropriate promoter, and an amplifiable marker gene under the controlof an appropriate promoter.

In those embodiments in which the DNA construct integrates into thecell's genome, it need include only the chimeric protein-encodingnucleic acid sequences. Optionally, it can include a promoter and anenhancer sequence, a polyadenylation site or sites, a splice site orsites, nucleic acid sequences which encode a selectable marker ormarkers, nucleic acid sequences which encode an amplifiable markerand/or DNA homologous to genomic DNA in the recipient cell to targetintegration of the DNA to a selected site in the genome (targeting DNAor DNA sequences).

C. Cell Culture Methods

Mammalian cells containing the chimeric protein-encoding DNA or RNA arecultured under conditions appropriate for growth of the cells andexpression of the DNA or RNA. Those cells which express the chimericprotein can be identified, using known methods and methods describedherein, and the chimeric protein isolated and purified, using knownmethods and methods also described herein, either with or withoutamplification of chimeric protein production. Identification can becarried out, for example, through screening genetically modifiedmammalian cells displaying a phenotype indicative of the presence of DNAor RNA encoding the chimeric protein, such as PCR screening, screeningby Southern blot analysis, or screening for the expression of thechimeric protein. Selection of cells having incorporated chimericprotein-encoding DNA may be accomplished by including a selectablemarker in the DNA construct and culturing transfected or infected cellscontaining a selectable marker gene under conditions appropriate forsurvival of only those cells which express the selectable marker gene.Further amplification of the introduced DNA construct can be effected byculturing genetically modified mammalian cells under conditionsappropriate for amplification (e.g., culturing genetically modifiedmammalian cells containing an amplifiable marker gene in the presence ofa concentration of a drug at which only cells containing multiple copiesof the amplifiable marker gene can survive).

Genetically modified mammalian cells expressing the chimeric protein canbe identified, as described herein, by detection of the expressionproduct. For example, mammalian cells expressing chimeric protein inwhich the second domain is transferrin can be identified by a sandwichenzyme immunoassay in which the chimeric protein is captured on amicrotiter plate by binding to a monoclonal antibody specific for theLDL binding domain, and the bound chimeric protein is detected bybinding to an anti-human TF monoclonal antibody specific for the humanTF domain. Routine assay for the level of chimeric protein in mostgenetically modified mammalian cell types that otherwise do notsynthesize human TF can be performed by an ELISA which detects human TF(Example 6), as each chimeric protein expressed contains an assayable TFdomain. Alternatively, mammalian cells expressing the chimeric proteincan be identified by an LDL binding assay (Example 9). Further, they canbe identified using other methods known to one of ordinary skill in theart. (Sambrook, J. et al., Molecular Cloning; a Laboratory Manual, 2ded., Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. A.et al., eds., Current Protocols in Molecular Biology, Wiley, New York,1994.

D. Purification of Chimeric Proteins

The chimeric protein produced by genetically modified mammalian cellscan be isolated from the cells and purified or partially purified usingmethods known to those of skill in the art as well as by methodsdescribed herein. As described in Example 7, chimeric protein has beenobtained, from cells in which it was expressed, through a method basedon direct binding to both domains of the chimeric protein. The methodincludes two steps: one involving immunoaffinity binding to thetransferrin epitope involved in transferrin receptor binding and anotherinvolving ligand (LDL) affinity binding to the LDLR domain. To purifyLDLR ligand-binding domain transferrin chimeric protein, the method wascarried out as follows: Medium in which cells expressing the chimericprotein were cultured is separated from the cells and, generally,concentrated by tangential flow ultrafiltration. The resulting culturemedium, which is enriched or concentrated for the chimeric protein, iscontacted with an anti-human transferrin monoclonal antibody (anti-TFMAb) bound to a column matrix, which results in binding of all proteinsin the medium which contain a transferrin epitope. The bound protein iseluted from the immunoaffinity column and the resulting immunoaffinitypurified chimeric protein is subsequently subjected to a ligand-affinitypurification step, in which it is contacted with LDL indirectly bound toa column matrix through an anti-LDL antibody fixed to the column matrix,under conditions appropriate for LDL-LDLR binding. As a result, chimericprotein containing transferrin and an intact LDLR ligand binding domainis bound to LDL; the chimeric protein is eluted from the column to yielda highly purified preparation of the chimeric protein. As demonstratedin Example 7, this process resulted in separation of intact chimericprotein which contains transferrin and binds LDL. The method can bemodified to purify chimeric proteins in which the first domain is aligand binding domain other than the LDLR ligand binding domain and thesecond domain is a cell surface receptor ligand other than transferrin.

In one embodiment of the present method of purifying chimeric proteincomprising intact LDLR binding domain and transferrin, the purificationis carried out as follows: Culture medium from host cells expressing thechimeric protein is separated from the host cells and concentrated bytangential-flow ultrafiltration using a membrane with a molecular weightcut off of 100,000 daltons. In one experiment, the culture medium isconcentrated approximately 32-fold.

An anti-TF MAb is (e.g., HTF-14, Biodesign, Kennebunk, ME, isolated fromascites fluid as described in Example 7) bound to a solid support, suchas polystyrene beads (e.g., cyanogen-bromide CNBr!--activated Sepharose4B), to form an immunoaffinity column. The concentrated culture mediumis contacted with the solid-support-bound anti-TF MAb, by loading themedium onto an HTF-14 immunoaffinity column, and maintained in contactwith the anti-TF MAb under appropriate conditions and for sufficienttime for the transferrin domain in the chimeric protein in the medium tobind to the anti-TF MAb (i.e., for the chimeric protein to bind toMTF-14 through an interaction with the chimeric protein's transferrindomain). As a result, chimeric protein containing transferrin isnon-covalently bound to the solid support (e.g., to beads to whichHTF-14 is bound). The solid support-bound chimeric protein is subjectedto appropriate conditions (e.g., washing with 0.1M glycine, pH 2.3) toelute the chimeric protein, which is collected.

In the embodiment described in Example 7, the HTF-14 immunoaffinitycolumn containing bound chimeric protein was washed with 0.1M Tris-HCl,0.15M NaCl, pH 7.4 (TBS), and then eluted with 0.1M glycine pH 2.3. Twomilliliter fractions were collected into buffer of appropriate pH toneutralize the elution buffer and certain of the fractions were pooled.Analysis showed that this step resulted in an approximately 8,000-foldpurification of chimeric protein from the concentrated culture media.The resulting immunoaffinity purified chimeric protein was a mixturewhich contained, as described in Example 7, chimeric protein whichincludes transferrin and intact LDLR binding domains and chimericprotein which had been degraded by a serine protease and, thus, did notinclude intact LDLR binding domain. The second step in the purificationprocess is a ligand-affinity (LDL-affinity) based step which results inseparation of chimeric protein which comprises transferrin and intactLDLR binding domain from the chimeric protein which does not includeintact LDLR binding domain. In this step, the immunoaffinity purifiedchimeric protein mixture was loaded onto a ligand affinity columncontaining CNBr activated Sepharose beads to which human LDL (hLDL) wasbound (e.g., by means of polyclonal rabbit anti-human LDL antibodyimmobilized on the beads). The immunoaffinity purified chimeric proteinwas loaded onto the anti-LDL (LDL) ligand affinity column for sufficienttime and under appropriate conditions for LDL on the beads and LDLR inthe chimeric protein to bind, producing chimeric protein non-covalentlybound to the column. The column was washed with TBS and eluted with 20mM EDTA in TBS, pH 7.4. Fractions were collected and assayed, andfractions 2 and 3 were shown to contain the peak concentrations of theprotein.

E. In Vitro Characterization of Chimeric Proteins

Analysis of chimeric protein produced as described herein (see Example10) showed that it binds LDL in a divalent cation-dependent manner,which is a well-established property of the LDL-LDLR bindinginteraction. Further analysis showed that binding of the chimericprotein to LDL did not occur in acidic buffer conditions and that EDTAand acidic buffer were each effective in dissociating chimeric proteinbound to LDL (Example 10). It is also a well-established property of theLDL-LDLR binding interaction that LDL is released from LDLR in vivo inthe acidic endosomal compartment. This suggests that the chimericprotein may be able to act in a similar way in cells, which would resultin release of the LDL from the chimeric protein in the cell. Takentogether, these findings support the function of the chimeric proteinwith respect to binding to serum LDL and uptake into cells for furthermetabolism.

Additional analysis (Example 9) demonstrated that half-maximal bindingoccurs at an LDL concentration of approximately 3 nM, which iscomparable to that of purified LDLR in a solid-phase binding assay(Innerarity, T. L. et al., Meth. Enzymol., 129:542-565 (1986)) as wellas to the published K_(d) value for the dissociation of LDL from LDLR inhuman fibroblast cells. This supports the idea that the chimeric proteinhas a binding affinity for LDL which is comparable to the bindingaffinity of full-length, plasma membrane-bound LDLR for LDL.

The functional activity of chimeric protein in which the first domain isthe ligand-binding domain of LDLR and the second domain is transferrinis assessed as described in Examples 11, 13 and 14. To determine whetherchimeric protein can result in cellular uptake of LDL via thetransferrin receptor, a hepatic cell line, such as HepG2, is used. Asdescribed in Example 13, hepatic cells and LDL in vitro are exposed tochimeric protein and LDL. To determine whether chimeric protein can bindhuman LDL in culture medium and mediate uptake into hepatic cells viathe transferrin receptor, the ability of unlabeled LDL or unlabeledtransferrin to inhibit cellular uptake of labeled LDL (e.g., ¹²⁵ I-LDL)in the presence of chimeric protein is assessed, as described in Example13. Whether LDL taken up by the transferrin receptor is metabolized to acholesterol pool can be assessed as described in Example 13. Forexample, inhibition of cholesterol biosynthesis as a result of LDLuptake can be assessed by determining production of an enzyme, such as3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase),which is down-regulated (inhibited) by an oversupply of cholesterol in acell. Alternatively, determination of levels or activity of an enzyme,such as acyl-coenzyme A cholesterol acyltransferase (ACAT), whichincreases in response to increased cholesterol in cells, can be used toassess whether LDL is taken up by the transferrin receptor andmetabolized to cholesterol. Increased ACAT activity is indicative ofincreased cholesterol levels in the cell.

F. In Vivo Characterization of Chimeric Proteins

The anti-hypercholesterolemic effect of the chimeric protein can beassessed, as described in Example 14. Briefly, chimeric protein isadministered in an animal model system for human familialhypercholesterolemia, such as the LDLR-knockout mouse or the Watanaberabbit, and its effect on serum cholesterol levels is determined. Adecrease in serum cholesterol levels after administration of anappropriate amount of chimeric protein to an LDLR-knockout mouse or aWatanabe rabbit is indicative of the ability of the chimeric protein totransport cholesterol into cells.

G. Therapeutic Use of Chimeric Proteins

Chimeric proteins of the present invention are useful to enhance LDLtransport into cells, such as hepatic cells. Chimeric proteins, such asthose with the LDLR-ligand-binding domain fused to a transferrin domain,can be administered to an individual in whom LDL metabolism is to beenhanced. Chimeric proteins are administered in an appropriate carrier,which can be physiologic saline or water or mixed with stabilizers orexcipients, such as albumin or low molecular weight sugars. They areadministered using known techniques and by a variety of routes, such asby intramuscular, intravenous, intraperitoneal injection. Alternatively,genetically modified mammalian cells expressing chimeric protein can beimplanted in an individual. Non-immortalized cells (primary or secondarycells) and/or immortalized cells can be transfected. These include, butare not limited to fibroblasts, keratinocytes, epithelial cells (e.g.,mammary epithelial cells, intestinal epithelial cells), ovary cells(e.g., Chinese hamster ovary or CHO cells), endothelial cells, glialcells, neural cells, formed elements of the blood (e.g., lymphocytes,bone marrow cells), muscle cells, hepatocytes and precursors of thesesomatic cell types.

Immortalized cells can also be transfected by the present method andused for either protein production or gene therapy. Examples ofimmortalized human cell lines useful for protein production or genetherapy by the present method include, but are not limited to, HT1080,HeLa, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinomacells, 2780AD ovarian carcinoma cells, Raji (ATCC CCL 86) cells, Jurkat(ATCC TIB 152) cells, Namalwa (ATCC CRL 1432) cells, HL-60 (ATCC CCL240) cells, Daudi (ATCC CCL 213) cells, RPMI 10 8226 (ATCC CCL 155)cells and MOLT-4 (ATCC CRL 1582) cells. In cases where geneticallymodified immortalized cells are used for gene therapy, the cells may beenclosed within a semi-permeable barrier device which allows for thediffusion of the chimeric protein out of the device.

Preferably, cells (e.g., fibroblasts) to be used in the gene therapymethod of the present invention are obtained from the individual to betreated. The cells are modified by introduction of a DNA construct ofthe present invention, such as a DNA construct encoding a chimericprotein in which domain 1 is the ligand-binding domain of human LDLR anddomain 2 is comprised of an amino acid sequence derived fromtransferrin. The resulting genetically modified cells are expanded inculture and introduced into the individual. Cells expressing a DNAconstruct encoding such a chimeric protein can be produced as describedin co-pending U.S. patent application Ser. No. 07/787,840, entitled "InVivo Protein Production and Delivery System for Gene Therapy", byRichard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov.5, 1991), the teachings of which are incorporated herein by reference.The cells express and secrete the chimeric protein, which binds LDL andtransports it to cells bearing transferrin receptors on their surfaces.Transferrin receptor-mediated endocytosis of the chimeric protein-LDLcomplex would result in introduction of LDL into cells and lowering ofextracellular LDL levels. DNA encoding a chimeric protein in which thesecond domain is a cell surface receptor ligand other than transferrin,such as those listed above, can be introduced into mammalian cells.Genetically modified mammalian host cells which express and secrete thechimeric protein can be implanted in an individual, in whom the chimericprotein binds LDL. The resulting chimeric protein-LDL complex is boundto cells bearing the receptor for which the amino acid sequence of thesecond domain is a ligand and, through receptor-mediated endocytosis,enters the cells. Thus, circulating LDL levels would be reduced.

The number of genetically modified mammalian host cells implanted in anindividual is determined by the amount of chimeric protein to bedelivered and the level of its expression by the cells. The amountneeded by an individual will depend on considerations such as age, bodysize, sex, serum LDL level, and serum half-life of the chimeric protein.The required dose can be determined empirically or calculated based onthe pharmacodynamic properties established for the chimeric protein.

Other, related, chimeric proteins can be used to clear oxidized LDL,which represents a clinically significant fraction of the total LDL,from the blood. The early stages of atherosclerotic plaque formation arebelieved to occur when high levels of circulating LDL interact withendothelial cells in blood vessels, where oxidation of amine groups(e.g. on lysine and arginine residues ) on apoB-100 takes place by freeradical attack. Oxidation (including acetylation) of apoB-100 in LDLparticles is known to result in loss of affinity for the LDL receptor.Oxidized LDL is not cleared by the liver, but instead accumulates in thecirculation until it can interact with the scavenger receptor (AcLDLR;previously known as the Acetyl LDL Receptor), which is predominantlyfound on macrophages. Macrophages taking up large quantities of oxidizedLDL become bloated with lipid vacuoles and are known as "foam cells".Foam cells with cell-surface determinants for adhesion to endotheliumare believed to play a role in the early development of atheroscleroticplaques and indeed are found in plaques at various stages.

A chimeric protein containing a scavenger receptor ligand-binding domainfused to human transferrin (AcLDLR/TF) is therapeutically useful bybinding to and removing oxidized LDL via transferrin receptors in theliver. In addition, since the chimeric protein can be supplied at highconcentration, it can function as a competitor of the macrophage AcLDLR,such that most oxidized LDL will be bound to AcLDLR/TF, rather thanbound to AcLDLR on macrophages. In this way, oxidized LDL accumulationin macrophages can be reduced and thus the numbers of foam cells foundin cardiovascular endothelium will also be reduced, thereby interferingwith early-stage atherosclerotic plaque formation.

More generally, other chimeric proteins are useful therapeutically toreduce levels of other biochemical substances associated with certaindisease states. The high-affinity ligand-binding domain of chimericproteins need not be restricted to those of known receptor molecules(e.g. LDLR or AcLDLR), but may also include other types of proteins withhigh binding affinity for protein ligands or small molecules. Moleculeswith the antigen binding properties of antibodies (for example, singlechain antibodies) or other proteins having reversible binding activitiescan be used to construct chimeric protein-encoding sequences with humantransferrin or with ligands which bind to other cell receptor ligands.

In one embodiment the chimeric protein has high-affinity bindingspecificity for apolipoprotein E4 (apoE4), but not to apoE3 or apoE2.Approximately half of all cases of Alzheimer Disease are associated withspecific allelic forms of apoE, and the E4 allelic form of apoE is foundto accumulate in amyloid fibrils in the brain and is a risk factor forthe disease. The apoE2 and apoE3 alleles are not associated withincreased risk and might play a role in disease resistance. A chimericprotein able to reduce apoE4 levels may be used therapeutically to slowthe development of Alzheimer Disease. By the methods described herein,chimeric proteins containing an apoE4 binding domain could be fused withtransferrin to produce a molecule which could remove apoE4 from thecirculation and ultimately reverse the accumulation that occurs inperipheral tissues.

Diagnostic Use of Chimeric Proteins

Chimeric proteins are also useful diagnostic reagents in biochemicalassays. For example, chimeric proteins can be used to determine thequantity of a selected substance, such as LDL, in a biological sample(e.g., cell lysates, blood, lymph, urine, water or milk). The biologicalsample to be analyzed is processed, if needed, to render the selectedsubstance available for binding to the first domain of an appropriatechimeric protein (e.g., for LDL, one in which the first domain is theligand-binding domain of human LDLR) and contacted with the chimericprotein under conditions appropriate for binding of the first domain andthe selected substance. The chimeric protein may be generally bound to asolid surface, such as a microtiter plate, polymeric beads or othersurface in such a manner that it remains bound to the surface underconditions used for binding of the selected substance to the firstdomain of the chimeric protein. If the selected substance is present inthe biological sample, it is bound to the chimeric protein and theresulting selected substance-chimeric protein complex is detected usingknown means (e.g., using an antibody which binds the selected substanceand is covalently linked (conjugated) to an active enzyme or radioactivenuclide. The activity of the enzyme is monitored, for example, bymeasuring cleavage of a chromogenic or fluorogenic substance.) Chimericproteins can detect a substance in a direct assay, with a high degree ofspecificity in a convenient format, such as in a microtiter plateformat. In one example presented herein, LDL is quantified by binding toLDLR/TF chimeric protein bound to a microtiter plate. The resultingbound LDL is detected by reaction with an anti-LDL antibody.

In other embodiments, chimeric proteins can substitute for antibodies inELISA assays. For example, LDL either directly or indirectly bound to aplate can capture a chimeric protein with an LDLR ligand-binding domain.This captured chimeric protein is then detected by reaction with anHRP-conjugated antibody directed against the second domain of thechimeric protein, for example, an anti-TF antibody can react with the TFdomain when an LDLR/TF chimeric protein is used. As another example,chimeric proteins which bind to components of the human immunodeficiencyvirus (HIV) may be used to detect the presence of the virus in complexbiological samples, such as blood or tissue specimens.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLE 1 Construction of a Plasmid Encoding an LDLR/TF Chimeric ProteinWith Amino Acids 1-374 of Human LDLR Fused to Amino Acids 20-698 ofHuman Transferrin

Described in this example is assembly of a gene encoding an LDLR/TFchimeric protein with amino acids 1-374 of mature human LDLR fused toamino acids 20-698 of human transferrin and construction of an LDLR/TFexpression plasmid. ##STR1##

Oligonucleotides 1-4 were utilized in polymerase chain reactions togenerate a fusion fragment in which a cDNA sequence corresponding toexons 1-8 of the human LDL receptor were fused to a cDNA sequenceencoding human transferrin. First, oligonucleotides 1 and 2 were used toamplify the 522 bp portion of the LDL receptor cDNA from pLDLR2 (ATCC#39966). Next, oligonucleotides 3 and 4 were used to amplify the 372 bpfragment comprised of transferrin sequences using plasmid TfR27A (ATCC#53106) as a template. Finally, the two amplified fragments were mixedand further amplified with oligonucleotides 2 and 3 to generate thefinal 834 bp fusion fragment. The PCR fusion joined LDL receptorsequences at valine 374 (numbered relative to the sequence of the matureLDLR protein) to valine 20 of transferrin sequences. This fusionfragment was digested partially with BamHI and completely with EcoRI.Analysis of the published DNA sequences of the LDLR cDNA (Genbankaccession number K02573) and the TF cDNA (Genbank accession numberM12530) predicts a 734 bp fragment from such a digestion of thePCR-generated fusion fragment. The 734 bp product was gel purified forcloning.

In order to construct a complete fusion between the first 374 aminoacids of the human LDL receptor and DNA sequence encoding amino acids 20to 698 of human transferrin, two intermediate plasmids were constructed.First, the entire transferrin cDNA was excised from TfR27A by partialdigestion with PstI. A 2.3 kb fragment containing the TF cDNA wasligated to PstI digested pBSIISK+(Stratagene, La Jolla, Calif.). Theligation mixture was transformed into E. coli and a clone containing asingle insert of the 2.3 Kb transferrin cDNA was isolated and designatedpBSIITF. Next, pBSIITF was digested with EcoRI and SacI to generate a1.4 kb fragment corresponding to the transferrin cDNA sequence. Thisfragment was gel purified, and ligated to EcoRI and SacI digestedpLDLR2. The ligation mixture was transformed into E. coli and a singleclone containing the 1.4 kb transferrin cDNA joined to LDL receptorsequences at the EcoRI site was isolated and designated pLT1.5. A 574 bpfragment of the transferrin CDNA was then gel purified from digestion ofTfR27A with EcoRI and BamHI. This fragment and the 734 bp LDLreceptor/transferrin fusion fragment (generated by EcoRI and partialBamHI digestion of the PCR product as described above) were ligated toEcoRI digested pLT1.5. The ligation mixture was transformed into E. coliand a single clone was isolated containing sequence encoding the first395 amino acids of the human LDL receptor (374 amino acids of the matureprotein with a 21 amino acid signal peptide) fused to the entire maturetransferrin coding sequence (amino acids 20-698). This plasmid wasdesignated pLDLrTF1.

To construct a plasmid useful for expression of the chimeric protein inmammalian cells, the chimeric protein coding sequences were isolatedfrom the plasmid pLDLrTF1 following digestion with SmaI and EcoNI. Thecohesive end resulting from EcoNI digestion was made blunt using theKlenow fragment of E. coli DNA polymerase. The digested DNA waselectrophoresed on a 1% low-melting agarose gel and the 3.4 kb DNAfragment corresponding to the chimeric cDNA was extracted. This fragmentwas ligated to the plasmid pEF-BOS (Mizushima, S. and Nagata, S.,Nucleic Acids Res. 18:5322 (1990)) that had been gel purified followingXbaI digestion and Klenow treatment. Plasmid pEF-BOS utilizes thepromoter sequences from the elongation factor-1a (EF-1a) gene. CompetentE. coli were then transformed with this ligation mixture. Transformantswere screened by restriction enzyme analysis, and one clone with thedesired orientation of the LDLR/TF fusion gene in plasmid pEF-BOS (theorientation in which the coding sequence extends 3' from the EF-1apromoter) was isolated and designated pEFBOS/LDLrTF1.S (FIG. 2). Thecomplete nucleotide sequence of the LDLR/TF fusion gene in plasmidpEFBOS/LDLrTF1.S is shown in FIG. 3.

EXAMPLE 2 Construction of a Plasmid Encoding an LDLR/TF Chimeric ProteinWith Amino Acids 1-710 of Human LDLR Fused to Amino Acids 20-698 ofHuman Transferrin

This example describes assembly of a gene encoding an LDLR/TF chimericprotein with amino acids 1-710 of human LDLR fused to amino acids 20-698of human transferrin and the construction of an LDLR/TF expressionplasmid. In this example of an LDL receptor-transferrin (LDLR/TF)chimeric protein, the fusion junction follows the valine codon atposition 731 of the mature LDLR polypeptide. The plasmid pLDLR2 obtainedfrom the ATCC (ATCC #39966) contains the cDNA sequences for the humanLDL receptor (LDLR). The cDNA sequence was inserted into plasmid pBS(Stratagene, La Jolla, Calif.) by the following steps to facilitate theconstruction of a chimeric gene. pLDLR2 was digested with EcoNI and theends were made blunt by treatment with the Klenow fragment of E. coliDNA polymerase. The treated DNA was then digested with BglII. The 1,735bp EcoNI-BglII fragment containing the 5' 1,721 bp of the LDLR CDNA aswell as 5' untranslated flanking sequences was isolated. A 976 bp DNAfragment containing the remaining 925 bp of the cDNA and 3' untranslatedflanking sequences was isolated following digestion of pLDLR2 with BglIIand NaeI. Plasmid pBS was linearized by digestion with EcoRV and theLDLR CDNA sequences was reassembled by ligation of the isolated 1,735 bp5' LDLR and 976 bp 3' LDLR fragments to EcoRV digested pBS using T4 DNALigase. After transformation of the ligation mixture into competent E.coli cells, individual bacterial clones were analyzed by restrictionenzyme analysis. One clone, with the properly assembled LDLR cDNA wasdesignated pBSL-1.

The human transferrin cDNA sequences were obtained from ATCC deposit#53106, clone TfR27A. The 2.3 kb transferrin cDNA sequences were excisedfrom TfR27A by digestion with Pst I and inserted into the Pst I site ofcloning plasmid pBSIISK+(Stratagene), resulting in the plasmid pBSIITF.

To prepare a DNA fragment containing the fusion junction between aminoacid 731 of LDLR (numbered relative to the amino acid sequence of themature protein) and amino acid 20 of TF, oligonucleotides LDLRTF710-1,-2, -3, and -4 were used in the polymerase chain reaction. ##STR2## Theplasmid templates were linearized in preparation for the PCR reactionsby digestion of pBSIITF with SalI and PBSL1 with NotI. Creation of thefragment containing the fused coding sequences was performed in twosteps. The first step consisted of a reaction mix containing 10 μl ofVent DNA polymerase (New England Biolabs, Beverly, Mass.), 1 ng each oflinearized pBSIITF and pBSL1 DNA, 12 μl of 2.5 mM dNTP's, 0.3 μl ofoligos LDLRTF710-2 and -4, 1 μl oligos LDLRTF710-1 and -3, and H₂ O tobring the final volume to 100 μl. The 1.4 kb fusion fragment resultingfrom this PCR amplification was further amplified with oligonucleotidesLDLRTF710-2 and -4. The amplified 1.4 kb fragment was gel purified anddigested with EcoNI and EcoRI and ligated to a 2.18 kb SalI-EcoNIfragment containing the LDLR sequences from pBSL1, a 1.55 kb EcoRI-XbaIfragment from pBSIITF (containing TF cDNA sequences) and SalI-XbaIdigested pBSIISK (Stratagene, La Jolla, Calif.). The ligation mixturewas used to transform competent E. coli cells. One clone with thecorrectly assembled fragments from the 4-way ligation was designatedpLDLRTF-710.

For expression of the chimeric cDNA in mammalian cells, the chimericcDNA of pLDLRTF-710 was subcloned into expression plasmid pEF-BOS(Mizushima, S. and Nagata, S., Nucleic Acids Res. 18:5322 (1990)) whichutilizes the promoter sequences from the elongation factor-1a (EF-1a)gene. pEF-BOS was digested with XbaI to remove the 450 bp stufferfragment. The SalI site lying at the junction between pBSIISK+and LDLRsequences of pLDLRTF-710 was modified by addition of an XbaI linker.Digestion of this modified fragment with XbaI generates a 4.4 kb XbaIfragment containing the LDLR/TF fusion gene. The 4.4 kb XbaI fragmentwas purified and ligated to XbaI digested pEF-BOS. The ligation mixturewas used to transform competent E. coli cells. One clone with thecorrect orientation of the LDLR/TF fusion gene XbaI fragment in plasmidpEF-BOS (the orientation in which the coding sequence extends 3' fromthe EF-1a promoter) was designated pEFBOS-LDLR/TF-710 (FIG. 4). Thecomplete nucleotide sequence of the LDLR/TF fusion gene in plasmidpEFBOS-LDLR/TF-710 is shown in FIG. 5.

EXAMPLE 3 Transfection of Mammalian Cells with Plasmids Encoding LDLR/TFChimeric Proteins and Identification of Clones Expressing LDLR/TFChimeric Proteins

A. Transfection of Primary Human Skin Fibroblasts

In this example, normal skin fibroblasts derived from newborn humanforeskins are cultured in a medium consisting of DMEM (Cellgro 50-013),15% bovine calf serum (Hyclone), units/ml of each of penicillin andstreptomycin (Gibco 15070-014), and 2.25% Hepes buffer (Gibco15630-015). Growing cells are washed in electroporation buffer (137 mMNaCl, 6 mM glucose, 5 mM KCl, 0.7 mM Na₂ HPO₄, 1 mg/ml acetylated BSASigma B-2518!, 20 mM Hepes buffer, pH 7.3) and resuspended inelectroporation buffer at 6 million cells per ml. Plasmid DNA (100 μgtotal) is added to an electroporation cuvette (BIO-RAD 165-2085) in avolume less than 50 μl, followed by addition of 0.5 ml of cellsuspension (3 million cells total).

Plasmid DNA consists of an equimolar mixture of the plasmid encoding thechimeric protein (in this example 61.2 μg of pEFBOS/LDLrTF1.S (8.66 kb)see Example 1) and the plasmid carrying a dominant-selectabledrug-resistance marker, in this example 38.8 μg of plasmid pSV2neo (5.50kb; ATCC #37149). Plasmid pSV2neo is used to provide a positiveselection for stably transfected cells, based upon resistance to thedrug G418.

The cuvette is subjected to an electric pulse (250 volts, capacitancesetting of 960 μFarad), permitting efficient entry of DNA into cells.Cells are diluted and plated into tissue culture dishes containing thesame growth medium having, in addition, 0.4 mg/ml of G418 (GENETICIN®,Gibco 860-1811). Only cells stably transfected with either pSV2neo orboth pSV2neo and pEFBOS/LDLrTF1.S are able to form colonies in thismedium. Cells that fail to integrate pSV2neo into their genomes arekilled by this drug and do not form clones. Other co-transfectingplasmids conferring dominant selectable drug resistance may be used.

After 2-3 weeks of incubation, G418-resistant cell clones are identifiedvisually and transferred to multi-well plates for further growth in thedrug-containing medium. Isolated drug-resistant transfectant clones arethen screened for co-expression and secretion of the chimeric proteinderived from the chimeric protein-encoding plasmid, in this examplepEFBOS/LDLrTF1.S.

To identify chimeric protein-expressing clones, conditioned medium fromeach clone is tested for the presence of the chimeric protein in anassay based on detecting, in this example, the cellular receptor liganddomain, which, in this example, is human transferrin. A sandwich enzymeimmunoassay for human transferrin (TF ELISA; see Example 6) is used todetect chimeric protein in the conditioned medium. Clones positive forLDLR/TF expression are cultured separately and analyzed quantitativelyfor levels of chimeric protein expression.

In one experiment, 206 transfected clones growing in medium containing0.4 mg/ml G418 were isolated with the aid of cloning rings andtransferred to 96-well culture dishes. After a growth period,conditioned media was tested by TF ELISA for the presence of TF antigenand 22 were found to be positive. Negative control cultures (clones ofuntransfected cells growing in the absence of G418 or G418-resistantclones isolated after transfection with pSV2neo) did not expressdetectable human TF antigen. 16 of the 22 positive clones were expandedfor further analysis. Cultures in T-25 flasks were grown tonear-confluence and exposed to fresh culture medium. After 24 hours, themedium was isolated for quantification of chimeric protein levels by TFELISA. The cells were then trypsinized and cell counts were determinedusing a Coulter Counter. The total amount of chimeric protein inconditioned media from each flask was calculated (in nanograms (ng)transferrin equivalents TF_(eq) !) and divided by the total number ofcells in each flask, resulting in a quantitative measure of expressionrate, which can be expressed as ng TF_(eq) per 10⁶ cells per day.Chimeric protein expression levels in 15 of the expressing clones weredetermined to be: 2, 7, 11, 21, 49, 52, 90, 93, 99, 131, 175, 193, 200,206, and 231 ng TF_(eq) per 10⁶ cells per day.

B. Transfection of Chinese Hamster Ovary (CHO) Cells

CHO cells are transfected with the chimeric protein expression plasmid,in this example pEFBOS/LDLrTF1.S, by the calcium phosphate precipitationprocedure (Graham, F. L., and van der Eb, A. J., Virology, 52:456(1973); Chu and Sharp, Gene, 13:197 (1981)). A co-transfecting dominantselectable marker plasmid, pSV2dhfr, is also used for clonal selectionof stably transfected cells, based on complementation of a nonfunctionaldihydrofolate reductase (dhfr) gene in the host CHO cell line,DUKX-B11(in Chasin, L. and Urlaub, G., Proc. Natl. Acad. Sci. USA,77:4216 (1980)). In the experiments described below, the dhfr gene inplasmid pSV2dhfr was used (S. Subramani et al., Mol. Cell. Biol.,2:854-864 (1981)), in which dhfr is expressed from an SV40 promoter.

Cells from DUKX-B11 are cultured in a complete medium consisting of aMEM(free of ribonucleosides and deoxyribonucleosides, Sigma M-4526), 10%fetal bovine serum (Hyclone A-1111), 4 mM L-glutamine (Gibco 25030-016),50 units/ml each of penicillin and streptomycin (Gibco 15070-014), 15μg/ml L-proline (Sigma P-4655), 10 μg/ml adenosine (Sigma A-4036), 10μg/ml thymidine (Sigma T-1895), and 10 μg/ml deoxyadenosine (SigmaD-8668).

Cells growing in a T75 flask at approximately 60-70% confluence in 20 mlof complete medium, were fed with 10 ml of fresh complete medium andincubated at 37° C. for 4 hours. Thirty minutes prior to the end of the4-hr period, a suspension of fine precipitates of calcium phosphate andplasmid DNA is prepared as follows:

Plasmid pEFBOS/LDLrTF1.S (50 μg) and pSV2dhfr (1 μg) were combined in atotal of 0.5 ml of TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.9)containing 0.25M CaCl₂. This solution was added dropwise to 0.5 ml ofanother solution containing 280 mM NaCl, 1.5 mM Na₂ HPO₄, 50 mM Hepes,pH 7.1. The 1 ml mixture was left undisturbed for 30 min. at roomtemperature to allow calcium phosphate precipitates to form. The 1 ml ofsuspension was added to the 10 ml of medium previously bathing the cellsfor 4 hours. The medium was swirled to disperse the particles and thecells were incubated at 37° C. for an additional 4 hours. The medium wasremoved by aspiration and the cells were contacted for 1 minute at roomtemperature with 5 ml of complete medium containing 20% glycerol. Theglycerol was subsequently removed by the addition of 15 ml of completemedium, followed by two additional washes of the cells with 20 ml eachof complete medium. The cells were then fed with 20 ml of completemedium and incubated at 37° C. for 24 hours, to allow chromosomalintegration and expression of the transfecting plasmid DNA. After 24hours, the cells were trypsinized and divided into 7 cultures (i.e.pools A-G) of equal size in DHFR selection medium.

DHFR selection medium provides a positive selection for only those cellsthat have taken up and expressed plasmid pSV2dhfr (with or withoutpEFBOS/LDLrTF1.S). Cells that do not express DHFR do not grow in thisselection medium. DHFR selection medium contains aMEM (see above), 10%dialyzed fetal bovine serum, 4 mM L-glutamine, 50 units/ml of each ofpenicillin and streptomycin and 15 μg/ml L-proline. This medium contains0.1% glucose and 0.22% sodium bicarbonate.

Pools A-G were grown to near confluence in DHFR selection medium for 17days, during which time fresh selection medium was replenished every 3or 4 days. Then, the cells in each pool were fed with 20 ml of freshselection medium, incubated for 24 hours, and the conditioned media wererecovered and tested for the level of chimeric protein by TF ELISA.

Bovine transferrin antigen contained in this selection medium does notcross-react with the antibodies against human transferrin that are usedin the TF ELISA. Moreover, the untransfected host CHO cell line,DUKX-B11, does not synthesize a secretable transferrin antigendetectable in the assay. Consequently, detection of any concentration oftransferrin antigen in conditioned media from transfected cellpopulations that is above the background level for the assay (1 ng/ml)constitutes evidence that the transfected population is expressing andsecreting a LDLR/TF chimeric protein.

Following the 17-day growth period in DHFR selection medium, mediaconditioned for 24 hours by cells from pools A through G containeddetectable levels of chimeric protein. In this example, pools A throughG produced 53, 52, 52, 79, 74, 75, and 33 ng TF_(eq) /ml, respectively,at the end of this 1-day conditioning period. Thus, initially all poolsproduce comparable but nonidentical levels of chimeric protein.Therefore, selection for expression of pSV2dhfr-encoded DHFR enzymeresults in co-expression of chimeric protein derived frompEFBOS/LDLrTF1.S, since a significant fraction of the cells integrateboth plasmids in an expressible form.

Cell populations A-G were subsequently treated with any one of a numberof well-established DHFR gene amplification protocols (Kaufman, R. J.and Sharp, P. A., J. Mol. Biol., 159:601 (1982)), which select forsubpopulations expressing higher levels of DHFR enzyme, which, in turn,is correlated with increased resistance to the toxic antifolate drugmethotrexate (MTX). In this example, pools were first selected forresistance to 20 nM MTX and then to 50 nM MTX (Sigma M-8407). At eachstep, the level of co-expression of secreted chimeric protein in eachrespective subpopulation is monitored by TF ELISA. Subpopulationsexhibiting increases in expression of chimeric protein product arelikely to contain certain high-expressing cells, which can be isolatedfrom the subpopulation by cell cloning.

In this example, subpopulations of pools E and F exhibited strongincreases in chimeric protein expression at 20 nM MTX and at 50 nM MTXas compared to the other pools. Cells from both pools E and F selectedat 50 nM MTX were subsequently cloned by limiting dilution methods; 9cell clones from pool E and 14 from pool F were identified as the bestproducers from a larger number of clones that had been isolated andtested by TF ELISA for chimeric protein production in multi-well cultureplates. These 23 clones, including the 2 highest from pool E (E77 andE117) and the 2 highest from pool F (F3 and F57) were evaluated further,with respect to potential for increased product output after additionalrounds of DHFR gene amplification (Example 4).

EXAMPLE 4 Identification of a Producer Cell Line and Assessment ofPotential Modifiers of Chimeric Protein Production

A. Identification of a producer cell line

Methotrexate (MTX)-resistant CHO cell lines derived from thedhfr-deficient CHO line DUKX-B11 were grown in DHFR selection medium(Example 3) with 100 riM MTX (Sigma M-8407). This medium contains 0.1%glucose. Some media also contained 2 mM sodium butyrate (Fluka 19364),or serine protease inhibitor aprotinin (Boehringer Mannheim 981-532) orPefablock SC (Boehringer Mannheim 1429-876).

From pool E, the two highest expressing clones (E77, which producedapproximately 1.5 μg TF_(eq) /ml over a 4-day period in 6-well platesand E117 which produced 1.1 μg TF_(eq) /ml over 4 days in 6-well plates)were cultured separately and amplified by selecting for resistance to100 nM MTX, to determine whether production of the chimeric proteinwould increase. All 9 clones from pool E were also combined into a newpool called pool H, which was subsequently amplified by selecting forresistance to 100 nM MTX. In the same manner, the two highest expressingclones from pool F (F3 and F57) were separately amplified by selectingfor resistance to 100 nM MTX, and all 14 clones from F were combinedinto a new pool called pool J, which was similarly amplified.

The 4 clones (E77, E117, F3, and F57) and pools H and J were evaluatedfor increased productivity (ng TF_(eq) per 10⁶ cells per day) in T75culture flasks as a function of increasing DHFR amplification.Production of chimeric protein was assayed by TF ELISA. The stability ofsuch productivity was also monitored by measuring product output duringsubsequent subculture at the same level of MTX resistance.

Results (see Table 1) show that clone E77 appeared to be the higheststable producer. Subsequent amplification to 200 nM and 500 nM MTX didnot result in higher productivity of chimeric protein. Similarly,selection for increased MTX resistance in pools H and J did not resultin selection for higher producing sub-populations.

Clone E77 appeared to grow well in 100 nM MTX and was chosen as theproducer cell line for roller bottles.

                  TABLE 1                                                         ______________________________________                                        Chimeric protein productivity (ng TF.sub.eq /10.sup.6                         cells/day) in CHO transfectant pools and clones as a                          function of MTX concentration and subculture round.                                 Sub-                                                                     MTX! culture                           Pool Pool                             (nm)  Round    E77     E117  F3   F57   H    J                                ______________________________________                                         50   1        1577    479   616  830   404  328                               50   2        1167    ND    552  608   ND   ND                               100   1        3554    800   620  1031  505  224                              100   2        1842    1532  347  604   381  187                              100   3        2011    729   641  1135  252  121                              100   4        1462    593   ND   ND    ND   ND                               200   1        1791    779   699  1045  404   90                              200   2        2000    728   855  948   327   97                              200   3        1598    928   975  546   510  100                              500   1        1577    957   855  ND    319   50                              500   2        ND      954   ND   ND    322   52                              ______________________________________                                         ND: not determined                                                       

B. Assessment of potential modifiers of chimeric protein production

The quantity and quality of chimeric protein production by producer cellline E77 was studied in T flasks and in roller bottles.

Previous immunoprecipitation experiments had shown that a small fractionof protein immunoprecipitated from the culture supernatants of humanfibroblasts transfected with plasmid pEFBOS/LDLrTF1.S retained the TFdomain but lacked an intact LDLR domain. The same was found to be trueof the chimeric protein secreted by the CHO cell line E77 (as well as inother clones and pools of CHO transfectants). The hypothesis that thissmaller product might result from the action of a specific endopeptidaseat a site within the LDLR domain was assessed by determining whether theaccumulation of this species could be inhibited by specific proteaseinhibitors or by using serum that had been heat inactivated. The serineprotease inhibitor aprotinin at 0.002% had a small effect in reducingthe relative abundance of this smaller species. Subsequently, a higheraprotinin concentration (0.5%) was tested.

Cells were grown to confluence in T75 flasks in 3 different media, allof which contained DMEM and 100 nM MTX. Medium A contained 10% dialyzedFBS (fetal bovine serum) and no additions, medium B contained 10% heatinactivated dialyzed FBS, while medium C was the same as medium A exceptfor the addition of 0.5% aprotinin. Cultures were fed with media A, B orC, respectively, and incubated. Small samples of conditioned media wereremoved at one and two days after feeding and incubated for 7 days at37° C. After incubation, each sample was electrophoresed on anon-reducing SDS/polyacrylamide gel, transferred to nitrocellulose, anddetected by binding to horse radish peroxidase (HRP) conjugated sheepanti-human TF.

The results showed that the smaller species accumulated after 2 days inmedium A, and was present at low abundance at all three conditions afterone day. Feeding with medium B (heat-inactivated serum) did not preventthis in vitro proteolysis, consistent with the view that the proteolyticagent was not a heat-inactivatable component of the serum. The additionof 0.5% aprotinin (medium C), however, significantly reduced the extentof proteolysis. This supports the idea that degradation of thefull-length chimeric protein, observed during the conditioning of mediaby confluent cultures, is caused by a secreted serine protease withspecific endopeptidase activity.

Inhibition of serine protease activity was also achieved using anotherinhibitor (Pefablock SC; PB). Increasing concentrations of PB in culturemedium results in decreasing relative abundance of the 100 kddegradation product, when 3-day conditioned media were analyzed bywestern blotting. Use of 0.6 mM PB appears to have a very strongsuppressive effect, although 0.3 to 0.5 mM PB also significantly reducesdegradation.

EXAMPLE 5 Roller Bottle Production of LDLR/TF Chimeric Protein

Initial attempts at scaling up chimeric protein production involvedusing 20 expanded surface (1450 cm²) roller bottles (Falcon 3069) with250 ml of medium per bottle, each rotating at 0.3 revolutions perminute. E77 cells were grown to confluence and fed and harvested weekly(Example 4). The 20 bottles were divided among 3 treatment groups.Roller bottles RB8 through RB12 were fed with 100 nM MTX medium. RB3-RB7were fed with medium lacking MTX. RB13-RB22 were fed with 100 nM MTXmedium, but had been seeded with a subclone of E77 called SC7.

Overall, 48.5 liters were produced which contained 96 mg TF_(eq) or 149mg of chimeric protein with an average yield of 3.1 mg of chimericprotein per liter. RB13-RB22 had the lowest average chimeric proteinconcentration of 2.2 mg/liter, while the groups RB3-RB7 and RB8-12produced an average of 3.3 mg/liter and 3.8 mg/liter, respectively. Fromthese results it would appear that removal of the MTX selective agentdid not increase yield. All 48.5 liters of conditioned media saved fromRB3-RB22 were pooled and concentrated for subsequent purification.

EXAMPLE 6 TF ELISA

In the sandwich enzyme immunoassay for detecting the human TF antigencontained within the chimeric protein (TF ELISA), MAb HTF-14 (BiodesignH61016M) was used to coat plates at 1:1000 in 1X PBS (Gibco 310-4200AJ).Human apo-TF (SIgma T-1147) in ELISA blocking buffer (PBS, 2% BSA, 0.05%Tween-20) was used as a standard. Human holo-TF (iron saturated)exhibited the same standard curve as apo-TF (iron-free). The enzymeconjugate for detection was horseradish peroxidase (HRP)-sheepanti-human TF (Biodesign K90070P) used at 1:5000 in ELISA blockingbuffer. The HRP substrate was OPD (Dako S-2000), prepared by dissolving8 mg OPD in 12 ml of 0.1M citric acid-phosphate buffer, pH 5.0, with0.0125% H₂ O₂.

The TF ELISA effectively detects the molar concentration of chimericprotein, based on comparison with the human apo-TF standard. Themolecular weight of the protein component of the chimeric protein (116.3kilodaltons) is 55% larger than the that of human TF (75.1 kd). Assumingthat the chimeric protein has the same affinity for MAb HTF-14 and forHRP-sheep anti-human TF as does the human TF standard, then theconcentration of chimeric protein can be expressed as a value equivalentto nanograms of TF equivalents per ml (i.e., ng TF_(eq) /ml). Thus, inthis case the actual concentration of chimeric protein on a ng/ml basisis approximately 55% higher.

EXAMPLE 7 Purification of LDLR/TF Chimeric Protein

The following materials and methods were used in this example. Forpurification of IgG from MAb HTF-14 ascites fluid, Goat anti-mouse IgGagarose beads (Hyclone EK-4081) were used. Mouse IgG was detected withHRP-conjugated Sheep anti-mouse IgG (Cappel 55565). HTF-14 IgG wasreacted with cyanogen bromide-activated sepharose to covalently bind theantibody to the sepharose beads for immunoaffinity (IA) purification ofTF epitope-containing chimeric protein species. For IA chromatographybased on LDL binding, affinity-purified rabbit anti-human LDL(Biomedical Technologies BT-905) was first reacted covalently withcyanogen bromide-activated sepharose, and then, in turn, was used tononcovalently immobilize purified human LDL (Biomedical TechnologiesTB-903). Rabbit IgG was detected with HRP-conjugated goat anti-rabbitIgG (Cappel 55689).

A. Purification by Anti-TF immunoaffinity chromatography

1. Isolation of IgG from MAb HTF-14 ascites fluid

The anti-transferrin monoclonal antibody HTF-14 was isolated fromascites fluid (Biodesign, H61016M, subclass IgGI) using a MonoclonalAntibody Affinity Isolation System (Hyclone, EK-4081). This systememploys as antibody anti-mouse IgG-coated agarose beads to bind andseparate the monoclonal from the ascites fluid. The antibody was elutedin a single step with 50 ml of 30 mM acetic acid containing 85% NaCl anddripped into solid sodium borate for a final concentration 0.1M sodiumborate, 30 mM acetic acid, 85% NaCl. The pools were immediately placedinto 12-14 kd cut-off dialysis tubing and dialyzed overnight at 4° wasthen frozen at -20° C. The pools were thawed, combined and concentratedwith Amicon Centriprep 50 to 12.5 ml at 4.36 mg protein/ml. Fifty-fourmilligrams of anti-transferrin monoclonal antibody was purified from 112ml of ascites fluid. The yield on this step of the purification was 99%of the available antibody.

2. Immobilization of HTF-14 to cyanogen bromodeactivated sepharose

The purified monoclonal anti-human transferrin antibody (HTF-14) wascovalently bound to Cyanogen-Bromide activated Sepharose 4B (CN-Br 4B)(Pharmacia). Twelve grams of support was swelled to 37 ml in 0.01N HCl.The beads were the washed with 10 volumes of 0.1M NaHCO₃ pH 8.3containing 0.5M NaCl. The antibody solution, HTF-14 at 4.36 mg/ml in0.1M NaHCO₃ pH 8.3 containing 0.5M NaCl, was added and allowed to reactwhile shaking gently on a motorized rotating platform for 20 hours at 4°C. Free amine groups were blocked via the addition of 30 ml of 1Methanolamine pH 8.5 for 3 hours with rotation.

3. Binding and elution of chimeric protein from concentrated conditionedmedia pool

The support was degassed and poured into a 50 ml Pharmacia Econo-column.It was equilibrated in TBS pH 7.4. The culture supernatant hadpreviously been concentrated 32-fold (48.5 liters to 1.5 liters). It wasre-assayed and shown to contain 62.3 mg of transferrin equivalents and1,044 grams of total protein. This culture supernatant was spun at10,000 rpm and filtered through a 0.22 μM filter. It was then loadeddirectly onto the HTF-14 immunoaffinity column at 100 mls/hour withcycling for 40 hours. The column was washed with 10 column volumes ofTBS pH 7.4. The column was then eluted with 0.1M glycine pH 2.3. Two mlfractions were collected into tubes containing 1 ml of 2M Tris-HCl pH8.5, to neutralize the elution buffer. Fractions 7-30 were pooled. Itwas determined that the pool contained 26.4 mg of chimeric protein (bothundegraded and protease degraded fractions) in 54 mg of total protein,corresponding to a purity level of 49% and a 8,193-fold purification inone-step from culture media.

B. LDL ligand-affinity chromatography

1. Immobilization of rabbit anti-human LDL to cyanogen bromide-activatedsepharose

Five milligrams of polyclonal rabbit anti-human LDL antibody (BiomedicalTechnologies, BT-905) was bound to Cyanogen-activated Sepharose 4B(CN-Br 4B) (Pharmacia). The rabbit anti-hLDL was dialyzed into 0.1MNaHCO₃ pH 8.3 containing 0.5M NaCl. 350 μg of support was swelled in0.01N HCl. The support was then washed with 10 volumes of 0.1M NaHCO₃ pH8.3 containing 0.5M NaCl. The antibody solution was added and reacted bygentle mixing for 24 hours at 4° C. Free amine groups were then blockedby the addition of 2 ml of 1M ethanolamine pH 8.5 for 1 hour with gentlemixing. 99% of the available antibody bound to the column, producing a 1ml anti-hLDL column with 5 mg of anti-hLDL covalently bound.

2. Binding of LDL

Ten milligrams of LDL (Biomedical Technologies, BT-903) at 5 mg/ml in 50mM Tris, 0.15M NaCl and 0.3 mM EDTA was obtained. It was dialyzed intoTBS containing 2 mM CaCl₂. The 10 mg of LDL was cycled 10 times over theanti-hLDL column at a concentration of 2 mg/ml. It was determined that3.2 mg of LDL remained in the flow-through, indicating thatapproximately 6.8 mg of hLDL was bound to the column.

3. Binding and elution of chimeric protein from IA pool

A 1 ml pool of concentrated immunoaffinity purified chimeric protein(purified on the anti-TF IA column) containing 138 μg chimeric proteinwas cycled 10 times over the 1 ml anti-hLDL/LDL ligand affinity column.Assuming a 1:1 ratio of undegraded protein to proteolytically cleavedprotein, this translates to approximately 67 μg of intact protein. Theflow-through contained 29 μg or 21% of the chimeric protein. The columnwas washed with 20 volumes of TBS pH 7.4, and then eluted with 20 mMEDTA in TBS pH 7.4. One ml fractions were collected. It was determinedby absorbance at 280 nm that fractions 2 and 3 contained the peak ofprotein. These fractions were concentrated in an Amicon Centriprep 100filtration unit by centrifugation for 30 min at 1000 rpm. It wasdetermined that 10.2 μg of the loaded intact chimeric protein was elutedin the peak fractions.

EXAMPLE 8 Purification of LDLR/TF Chimeric Protein Utilizing ProteaseInhibition and Dissociation of Bound Bovine LDL

The previous example describes a purification protocol that isolatesundegraded LDLR/TF chimeric protein from roller bottle cultures of CHOproducer cell line E77, employing the following steps: 1) accumulationand concentration of conditioned media, 2) immunoaffinity chromatography(specific binding of the human transferrin domain of the chimericprotein to immobilized HTF-14 monoclonal antibody), and 3)ligand-affinity chromatography (specific binding of the human LDLRdomain of the chimeric protein to immobilized human LDL). While theprevious example describes a two step procedure for isolating LDLR/TFchimeric protein, the LDL ligand-affinity chromatography step isrelatively inefficient (i.e. only a fraction of the load protein isrecovered). This is probably due to the fact thatimmunoaffinity-purified chimeric protein contains bound cholesterol,likely in the form of bovine LDL particles derived from the dialyzedfetal bovine serum used during cell culture. Thus, human LDL binding maybe inhibited in a fraction of chimeric protein molecules due to boundserum-derived bovine LDL-cholesterol. The protocol presented in thisexample involves dissociation of bovine LDL from the chimeric proteinprior to binding to immobilized human LDL.

Chimeric protein is accumulated in culture medium containing the serineprotease inhibitor Pefablock® SC (added to fresh culture medium at aconcentration of 0.2 mM). This inhibitor has no significant effect onthe level of total chimeric protein produced, but it does increase therelative yield of intact LDLR/TF. Conditioned medium is concentrated andloaded onto an HTF-14 immunoaffinity column. Since LDL binding to thechimeric protein's LDLR domain is divalent cation-dependent, while thebinding of MAb HTF-14 to the chimeric protein's transferrin domains isdivalent cation-independent, the column containing bound chimericprotein is washed with EDTA to specifically elute bovine LDL bound tochimeric protein. Following the EDTA wash, chimeric protein is elutedwith 0.1M glycine (pH 2.3) as described in Example 7, with the resultbeing that the LDLR/TF product contains much less associatedcholesterol. This material is useful for in vitro and in vivoexperiments or for therapeutic use. Optionally, it may also be purifiedfurther by human LDL ligand-affinity chromatography to remove degradedproduct which has lost the LDLR binding domain.

In order to illustrate the difference between immunoaffinity-purifiedchimeric protein with bound bovine LDL (Example 7) andimmunoaffinity-purified chimeric protein depleted of bovine LDL (thisExample), chimeric protein produced with protease inhibitor is firstbound to the HTF-14 immunoaffinity column, eluted, and reapplied to theHTF-14 column. A total of 43.51 liters of conditioned medium produced inthe presence of 0.2 mM Pefablock® SC and containing 56.1 mg of chimericprotein is concentrated to a volume of 1.17 liters containing 50.8 mg ofchimeric protein (91% recovery), using a Pellicon Tangential-Flow FilterSystem fitted with a 100,000 dalton molecular weight cutoff filtercassette (Millipore). The concentrate (50.8 mg of chimeric protein, 90.7g of total protein) is applied to the HTF-14 column (87% binding) andeluted with 0.1M glycine (pH 2.3) as described in the previous example.Nearly all of the eluted chimeric protein is released in the first 24fractions. These fractions were pooled and contain 35.9 mg of chimericprotein and 82.6 mg of total protein, corresponding to a 776-foldpurification with 71% recovery. This eluted pool of chimeric protein(called IA1) was found to contain 2.9 mg of total cholesterol per mg ofchimeric protein.

24.4 mg of chimeric protein from pool IA1 was reapplied to the HTF-14column (99.9% binding). Elution with 20 mM EDTA did not releasesignificant amounts of chimeric protein (0.005% of total bound chimericprotein was released). Subsequent elution of chimeric protein with 0.1Mglycine (pH 2.3), pooling of fractions in the major elution peak, anddialysis of the pool against PBS yielded a pool containing a total of19.13 mg of chimeric protein (78% recovery). This eluted pool ofchimeric protein (called IA2) was found to contain only 0.13 mg of totalcholesterol per mg of chimeric protein. Thus, greater than 95% ofcholesterol bound to the chimeric protein was removed by washing thecolumn with 20 mM EDTA prior to glycine elution.

For further purification, the IA2 preparation of chimeric protein wasapplied to the human LDL ligand-affinity column in the presence ofdivalent cation, followed by elution with a molar excess of EDTA as inExample 7. The loaded material, depleted of bovine LDL, binds and eluteswith greater overall recovery as compared to the material described inExample 7, in which column binding and recovery was inhibited due tobovine LDL bound to the chimeric protein.

FIG. 6 shows LDLR/TF chimeric protein forms, produced in CHO cells, atvarious stages of immunoaffinity (IA) purification and analyzed bynon-reducing SDS/PAGE and Western blot analysis. Protein electroblottedto nitrocellulose is bound to peroxidase-conjugated anti-humantransferrin antibody, followed by chemiluminescent detection ofperoxidase. The major band is the LDLR/TF chimeric protein, while thesmaller, minor band represents a product that has lost the LDLR bindingdomain.

Lane 1 shows concentrated conditioned medium from CHO cells expressingLDLR/TF chimeric protein. Lane 2 shows LDLR/TF chimeric protein purifiedfrom CHO cell supernatants by the method of Example 7. The materialloaded is an aliquot of a pool of protein fractions eluted from the IA(MAb HTF-14) column. Lane 3 shows LDLR/TF chimeric protein pool of lane2 after dialysis against TBS. Lane 4 shows LDLR/TF chimeric proteinpurified from CHO cell supernatants by the method described in Example7. Concentrated conditioned medium was bound to and eluted from theanti-human transferrin immunoaffinity column, followed by binding to andelution from the human LDL ligand-affinity column. Note the depletion inthe lower band of immunoreactive material. Lane 5 shows LDLR/TF chimericprotein purified from CHO cell supernatants by the method described inExample 8, in which the step for dissociating bound bovine LDL wasincluded. Chimeric protein (IA1) from the pool shown in lane 3 was boundagain to the IA column and then depleted of bound bovine LDL by washingthe column in TBS containing 20 mM EDTA. Chimeric protein shown here(called IA2) represents a pool of protein fractions eluted from the IAcolumn after the EDTA washing step. The material in lanes 1, 2, 3, and 5was isolated from CHO cells grown in the presence of the serine proteaseinhibitor Pefablock SC.

EXAMPLE 9 Measurement of LDL Binding to LDLR/TF by an In Vitro BindingAssay

A microplate binding assay was developed in order to study the bindingaffinity of LDL for LDLR/TF. In this assay, human LDL is captured on theplate by chimeric protein bound to the plate via an anti-TF monoclonalantibody used to coat the wells. Human LDL is then detected by reactionwith an anti-LDL antibody. This assay is dependent on the chimericprotein containing both the TF and LDLR domains.

A 96-well ELISA microplate was coated with MAb HTF-14 (1:500 in 50 mMTris-HCl, 2 mM CaCl₂, pH 8.0) for 45 minutes at 37° C., after which theplate is washed 3 times in solution B (50 mM Tris-HCl, 2 mM CaCl₂, pH8.0, 0.5% BSA, 0.05% Tween-20). Wells were incubated for 45 minutes at37° C. in blocking solution (50 mM Tris-HCl, 2 mM CaCl₂₁ pH 8.0, 1% BSA,0.05% Tween-20). Blocking solution was removed and replaced with variousbinding media and incubated for 30 minutes at 37° C. Binding mediacontained human LDL in solution B at concentrations between 0.5 μg/ml(0.97 nM apoB-100) and 50 μg/ml (97 nM apoB-100), and, in addition,either chimeric protein at 1.55 μg/ml (13.3 nM), human transferrin at 1μg/ml (13.3 nM), or nothing added. The plate was washed 3 times withsolution B and then incubated for 30 minutes at 37° C. with a sheeppolyclonal Ab (IgG) specific for human LDL (1:1000 in solution B;Biomedical Technologies, Inc., BT-999). The plate was washed 3 timeswith solution B and then incubated for 20 minutes at 37° C. with aperoxidase-conjugated rabbit polyclonal Ab specific for sheep IgG(1:5000 in solution B; Cappel, 55814). The plate was washed 3 times withsolution B and then incubated for 5 minutes at 37° C. with theperoxidase substrate 1,2-phenylenediamine (0.67 mg/ml in 0.1M citricacid-phosphate, pH 5.0). The reactions were stopped by the addition ofH₂ SO₄ to a final concentration of 0.8N, after which the absorbances at490 nm were determined using a microplate reader. At each concentrationof LDL, the signal obtained in binding media containing humantransferrin was comparable to that obtained in the absence oftransferrin or chimeric protein, and this is considered to constitute abackground level of nonspecific binding. The mean absorbance due tononspecific binding at each LDL concentration was subtracted from thesignal obtained in the presence of chimeric protein to yield anabsorbance value representing specific binding of LDL to chimericprotein. This absorbance value was plotted versus LDL concentration, asshown in the FIG. 7. Assuming that the signal at 50 μg/ml representsmaximum binding, half-maximal binding was observed at approximately 1.6μg/ml (3.1 nM) of LDL.

EXAMPLE 10 Binding and Dissociation Properties of LDLR/TF ChimericProteins

The LDL:LDLR interaction has been characterized in great detail, andshown to require Ca⁺⁺ or other divalent cations. Additionally, bindingof the LDL ligand to the LDLR is dependent on pH, such that dissociationoccurs at low pH (J. L. Goldstein and M. S. Brown, Ann. Rev. Biochem.,46:897 (1977)). This latter characteristic is important physiologically,allowing release of LDL by the LDLR into the acidic, endosomalcompartment, and recycling of the receptor. Therefore, an in vitroassay, designed specifically to study the LDL:LDLR interaction, was usedto study the pH and cation independence of LDL binding to anddissociation from the LDLR/TF chimeric protein.

LDLR/TF chimeric protein produced and purified as described in Example 8was examined for its LDL binding properties. Specifically, the cationdependence of LDL binding to the LDLR domain of the chimeric protein wasstudied by examining binding in the presence and absence of EDTA. Thecation dependence of LDL binding to the LDLR domain of the chimericprotein was studied by examining the effects of post-binding washingsteps, performed in the presence and absence of EDTA. Similarly, the pHdependence of LDL binding to the chimeric protein was studied byexamining the effects of pH 8.0 versus pH 5.2 conditions during thebinding and post-binding wash steps, respectively. Decreased binding atlow pH would indicate that LDL may be released from the LDLR/TF:TFRcomplex in the acidic environment of the endosome, permitting therecycling of the complex. A microplate binding assay was developed toexamine these effects and the results are shown in FIG. 8.

The microplate binding assay detects the amount of chimeric proteinbound to LDL immobilized on an ELISA plate coated with an anti-LDLantibody. A 96-well microtiter plate was coated with 5 μg/ml sheepanti-human LDL antibody (Biomedical Technologies, Inc., BT-999) in 50 mMTris-HCl, pH 8.0, 2 mM CaCl₂ (solution A). After washing with solution Aplus 0.5% BSA and blocking with solution A plus 1% BSA at 37° C. for 30minutes. Sample wells that served as negative controls were not coatedwith the anti-human LDL antibody (no Ab). Unbound LDL was removed bywashing with solution A plus 0.5% BSA. Since LDL:anti-LDL binding isexpected to be insensitive to divalent cation concentration, certaincontrol wells were washed instead with solution A plus 0.5% BSA and 20mM EDTA (pre-wash in EDTA). Next, chimeric protein was added to allwells at a concentration of 1 μg/ml in solution A plus 0.5% BSA(untreated), and incubated at 37° C. for 30 minutes. Some wells weretreated with chimeric protein in solution A plus 0.5% BSA and 20 mM EDTA(binding in EDTA), while others were treated with chimeric protein in apH 5.2 buffer containing 25 mM sodium acetate, 150 mM NaCl, and 2 mMCaCl₂ (binding in pH 5.2). Sample wells were then washed with one ofthree solutions: 1) solution A plus 0.5% BSA (untreated), 2) solution A,plus 0.5% BSA and 20 mM EDTA (wash in EDTA), or 3) 25 mM sodium acetate(pH 5.2), 150 mM NaCl, 2 mM CaCl₂ (wash in pH 5.2). Wells were thenincubated with an HRP-conjugated sheep anti-human transferrin antibody(Biodesign K90070P, 1:5000 dilution in solution A plus 0.5% BSA),incubated for 30 minutes at 37° C., followed by thorough washing withsolution A, plus 0.5% BSA untreated). Some wells were washed withsolution A, plus 0.5% BSA and 20 mM EDTA (post-HRP wash in EDTA), whileothers were washed in 25 mM sodium acetate (pH 30 5.2), 150 mM NaCl, 2mM CaCl₂ (post-HRP wash in pH 5.2). The final wash step was followed bydevelopment with the substrate ortho-phenylenediamine for 5 minutes at37° C. After addition of 2N H₂ SO₄ to stop the reaction, the plate wasanalyzed for absorbance at 490 nm.

As seen in FIG. 8, the binding of chimeric protein to the plate(untreated) is entirely dependent on the presence of plate-bound LDL,since lack of coating antibody (anti-LDL) resulted in only a backgroundlevel of chimeric protein binding (no Ab). Moreover, EDTA did notsignificantly dissociate LDL bound to the coating antibody, asdemonstrated by the small effect on the final chimeric protein bindingsignal (pre-wash in EDTA).

Chimeric protein binding to plate-immobilized LDL was dramaticallyreduced to background or near-background levels if this binding step wascarried out in the presence of a 10-fold molar excess of EDTA over Ca⁺⁺(binding in EDTA) or under acidic conditions (binding in EDTA) or underacidic conditions (binding in pH 5.2). This shows that the chimericprotein:LDL binding interaction is dependent on the presence ofunchelated divalent cations and is pH-sensitive, similar to the LDL:LDLRbinding interaction observed in vitro and in vivo.

Following chimeric protein binding under permissive conditions,subsequent wash steps performed in either EDTA (wash in EDTA) or underacidic conditions (wash in pH 5.2) were effective in dissociatingchimeric protein from LDL to background or near-background levels. Theseresults suggest that the chimeric protein can release its bound LDLunder acidic conditions, as observed for LDLR in vitro and in vivo.

Following chimeric protein binding, washing, and binding to theHRP-conjugated anti-human transferrin antibody, subsequent wash stepsperformed in either EDTA (post-HRP wash in EDTA) or under acidicconditions (post-HRP wash in pH 5.2) were only partially effective indissociating chimeric protein from LDL. Thus, these results demonstratethat the effects of pH and EDTA are due to dissociation of LDL:chimericprotein complexes, and not to inhibition of binding of HRP-conjugatedanti-human transferrin antibody to the TF domain in pre-formedLDL:chimeric protein complexes. Furthermore, these results suggest thatbinding of the antibody to the TF domain of the chimeric protein has anallosteric effect on the LDLR domain (causing increased LDL affinity),or perhaps causes a steric hindrance towards dissociation reagents(causing increased stabilization of the LDL:chimeric protein complexunder dissociation conditions).

EXAMPLE 11 LDLR/TF Chimeric Proteins Expressed in Mammalian CellsContain Intact LDLR and TF Structural Domains

This example describes work demonstrating that a single LDLR/TF chimericprotein contains both LDLR and TF structural domains which, based on theknown specificities of the MAbs used, indicates that these structuralregions retain functional binding activity for LDL and TFR,respectively.

LDLR/TF chimeric proteins are expressed in transfected mammalian cellsfrom a fusion gene consisting of a 5' human LDLR cDNA sequence (374amino acid ligand-binding domain) fused in frame at its 3' end with theentire cDNA sequence encoding mature human transferrin. To demonstratethat the chimeric protein retains the structural features found in thenatural LDLR and TF proteins, we show here that the LDLR/TF chimericprotein species can be immunoprecipitated by two different MAbs: HTF-14(anti-human TF, Biodesign H61061M) and C7 (anti-human LDLR, Amersham RPN537).

MAb HTF-14 is known to react specifically with a conformational (andnon-reduced) epitope on human TF that maps at or near the TFR bindingsite. Binding of HTF-14 to TF inhibits TFR binding. The binding of MAbHTF-14 to LDLR/TF chimeric protein constitutes evidence that the TFdomain of the chimeric protein is largely intact and suggests that thisconformation in LDLR/TF will also have TFR binding activity. Example 12presents data demonstrating that the LDLR/TF chimeric protein does infact bind to human TFRS.

MAb C7 is known to react specifically with the ligand-binding domain ofhuman LDLR at or near the LDL (ligand)-binding site. The binding of C7to LDLR inhibits LDL binding and, conversely, the binding of LDL to LDLRinhibits C7 binding. The binding of MAb C7 to LDLR/TF chimeric proteinconstitutes evidence that the LDLR domain of the chimeric protein islargely intact and suggests that this site in LDLR/TF will also haveLDL-binding activity. Other experiments (Example 9) have shown that theLDLR/TF chimeric protein does in fact bind to human LDL.

One ml of conditioned media from normal human fibroblast clonestransfected with either pSV2neo (negative control) or co-transfectedwith pEFBPS/LDLrTF1.S and pSV2neo was first pre-cleared with goatanti-mouse IgG agarose beads (Hyclone EGK-1060) in TBS containing 1%NP-40, followed by the addition of either MAb HTF-14, MAb C7, or anunrelated MAb (anti-human factor IX, Hematologic Technologies, Inc.AHIX-5041). After antibody binding, antigen-antibody complexes wereprecipitated by addition of goat anti-mouse IgG agarose beads. The beadswere washed to remove unbound material and proteins were eluted from thebeads by boiling with SDS/PAGE sample buffer containing SDS. Eluateswere run on SDS/PAGE (8% gel), electroblotted to a supportednitrocellulose membrane filter (Schleicher and Schuell BA-S-85), and thefilter was blocked for several hours in TBS containing 0.05% Tween-20and 5% (w/v) nonfat dry milk. The blocked filter was treated withHRP-conjugated sheep anti-human TF (Biodesign K90070P), which detectsprotein species carrying a variety of human TF epitopes, including thatrecognized by HTF-14. Antibody binding is performed here in TBScontaining 0.05% Tween-20. After the binding step, the filter was washedseveral times in TBS with 0.05% Tween-20 followed by two washes withTBS. Following detection of the HRP conjugate with chemiluminescencereagents (Amersham RPN 2106), the film was exposed, processed, andscanned (FIG. 9). Lane 1 contains 1 ml of conditioned medium from thenegative control human fibroblast clone (SV2neo-transfected)immunoprecipitated with MAb HTF-14. There are no chimeric proteinspecies present. The material observed in lane 1 is not seen when 2%nonfat dry milk is used in the binding medium, and is thus not likely tobe related to the chimeric protein. Lane 2 contains 1 ml of conditionedmedium from a human fibroblast clone co-transfected withpEFBOS/LDLRrTF1.S and pSV2neo and immunoprecipitated with MAb HTF-14. Amajor protein species (approximately 116 kd) and one minor species(approximately 100 kd) is evident. The minor species is derived from themajor species by a specific serine protease cleavage event in the LDLRdomain. Lane 3 contains 1 ml of conditioned medium from the same humanfibroblast clone used in lane 2, but immunoprecipitated with MAb C7. Amajor protein species (approximately 116 kd) is evident which has thesame molecular size as the major product immunoprecipitated by MAbHTF-14. This indicates that a single LDLR/TF chimeric protein containsboth an LDLR domain (immunoprecipitable with C7) and a TF domain(Detectable with HTF-14 in the Western blot analysis). MAb C7 does notimmunoprecipitate the minor product seen in lane 3, which retains anintact TF domain but lacks an intact LDLR domain. Lane 4 contains 1 mlof conditioned medium from the same human fibroblast clone shown inlanes 2 and 3, but immunoprecipitated with a MAb specific for humanfactor IX, which is not known to bind to either LDLR or TF. As expected,the LDLR/TF chimeric protein is not immunoprecipitated by this antibody.

EXAMPLE 12 Binding of LDLR/TF Chimeric Protein to Transferrin Receptorson Human Hepatic Cells In Vitro

The LDLR/TF chimeric protein purified as described in Example 8 has beentested for functionality by its ability to bind to the transferrinreceptor (TFR) present on the surface of human hepatic cells. Asdescribed in this example, the LDLR/TF chimeric protein blocks TFbinding to cells, indicating that it binds the transferrin receptor onhuman hepatic cells. Hep G2 cells are useful for in vitro studies ofhepatocytes since they synthesize most of the proteins normally made byhepatocytes in normal human liver, including the TFR.

Hep G2 cells (ATCC number HB 8065) were grown in medium consisting of(DMEM, Cellgro 50-013), 10% fetal bovine serum (Hyclone A-1111), and 50units/ml each of penicillin and streptomycin (GibcoBRL 15070-014).Radiolabeled transferrin ligand was obtained from Amersham (IM 194) as(3- ¹²⁵ I!iodotyrosyl)-transferrin (human), at a specific activity of700-800 Ci/mmol. This ¹²⁵ I-TF is iron-saturated (i.e. holo TF) and wasdissolved in a binding medium containing 80% DMEM (Cellgro 50-013), 0.5%protease-free bovine albumin (Sigma A-3059), and 20% phosphate-bufferedsaline (PBS; GibcoBRL 14200-026). Inhibitors of radiolabeled TF bindingwere diluted in PBS and added to binding medium by substituting for thePBS component. In this example, chimeric protein and unlabeled holo TFwere tested for inhibitory activity.

Hep G2 cells were plated into wells of 6-well tissue culture plates (35mm diameter) at 2×10⁵ cells per well and grown for 3 days. Cells werethen washed one to two times with DMEM and subsequently incubated at 37°C. with 5 ml of DMEM for 30 minutes. This washing and incubation stephelps remove serum components introduced with the fetal bovine serum,including transferrin, that might otherwise be bound to TFR andinterfere with the binding assay. After the 30 minute incubation, theplates were placed on ice and the medium removed by aspiration. Eachwell received one ml of ice-cold binding medium and the plates wereagitated slowly by shaking or rocking at 4° C. for 2 hours at (thebinding period).

After the 2 hr. binding period, the plates were placed on ice and kepton ice during subsequent washing steps. To remove unbound label from thecells, each well was washed 3 times with PBS containing 0.5% BSA,followed by an additional 3 washes in PBS. The cells in each well werethen lysed with 1 ml of 1N NaOH and the lysates transferred to 12×75 mmpolystyrene tubes (Falcon 2052). The wells were washed with anadditional 1 ml of 1N NaOH, which was added to the tube containing theappropriate lysate. The amount of radioactive TF present in the lysateswas determined by quantification of gamma emission in a gamma counter(Beckman). Since placing the cells on ice at the conclusion of thebinding period blocks uptake of labeled TF into the cells, the gammaemissions counted reflect the amounts of labeled TF on the surface ofthe cells at the end of the binding period. Lysates were alsoneutralized and assayed for total protein content using the BCA ProteinAssay (Pierce 23225G), with bovine albumin used as a standard. The ratioof total radioactivity (expressed as counts per minute; cpm) to totalprotein content (in milligrams) gives a good estimate of ligand bindingcapacity.

The equilibrium dissociation constant (K_(d)) for TF binding to TFR inHep G2 cells is 7 nM (Trowbridge, I. S. et al., Biochem. Pharmacol.,33:925-993 (1984)). If unlabeled TF is present at 7 nM as a competitiveinhibitor of ¹²⁵ I-TF binding to TFR, ligand binding (in cpm/mg) will beinhibited approximately 50%. Furthermore, if any unlabeled ligand(present at a concentration of approximately 7 nM) can bind to TFR withan affinity comparable to that of TF (e.g. LDLR/TF), 50% inhibition ofbinding will be observed. At high concentrations of unlabeled ligand(e.g. 50- or 100-fold higher than the K_(d)), nearly all receptorbinding sites will be occupied and the only ligand bound to the cellsshould be that bound to nonspecific sites (i.e. sites other than theTFR). If an unlabeled ligand does not bind to TFR, no binding inhibitionwill be observed. Thus, human LDL, which does not interact directly withthe TFR, does not display inhibition of TFR binding to ¹²⁵ I-TF.

This ligand-binding assay has been used to show that LDLR/TF chimericproteins exhibit competitive inhibition of TFR binding to ¹²⁵ I-TF inHep G2 cells and therefore can bind specifically to the human TFR.

In one experiment, Hep G2 cells were incubated with either no inhibitoror holo TF or purified LDLR/TF chimeric protein purified from CHO cellsexpressing the LDLR/TF chimeric protein with amino acids 1-395 of humanLDLR fused to amino acids 20-698 of human transferrin (see Example 3)!to assess the inhibitory effect these proteins. Holo TF and chimericprotein were tested at concentrations of 5 nM, 50 nM, or 500 nM with 10μM FeCl₃ in an attempt to further saturate the iron-binding sites of TFor LDLR/TF chimeric protein. The results are shown in Table 2 (below):

                  TABLE 2                                                         ______________________________________                                        Binding (cpm bound per mg total protein in lysate)                            of .sup.125 I-holo TF.sup.a to Hep G2 cells.sup.b                                                                  500 nM/10                                INHIBITOR 0 nM    5 nM       500 nM  μM FeCl.sub.3                         ______________________________________                                        None      62,856  --         --      --                                       holo TF   --      33,602 (53%)                                                                             1,026 (2%)                                                                            1,160 (2%)                               LDLR/TF   --      73,982 (117%)                                                                            3,363 (5%)                                                                            2,724 (4%)                               chimeric protein                                                              ______________________________________                                         The numbers in parentheses denote percentage of counts bound relative to      binding in the absence of inhibitor (set at 100%).                            .sup.a 0.32 nM labeled TF                                                     .sup.b 35 mm wells seeded at 2 × 10.sup.5 cells                    

These results show that both holo TF and LDLR/TF chimeric proteins werepotent inhibitors of labeled TF binding. This ligand-binding assay thusdemonstrates that LDLR/TF chimeric proteins exhibit competitiveinhibition of TFR binding to ¹²⁵ I-TF in Hep G2 cells and therefore canbind specifically to the human TFR.

A similar assay can be used to show that chimeric proteins can also bindto TFR present on the surface of human fibroblasts and other cell types.

EXAMPLE 13 Uptake of LDL into Human Hepatic Cells In Vitro

The LDLR/TF chimeric protein purified as in the previous examples can betested for the ability to bind both to human LDL and to human TFR insuch a way as to enhance cellular uptake of LDL via a TFR-mediatedpathway. Moreover, the additional LDL uptake afforded by the chimericprotein can be inhibited by competition with TF.

To quantify LDL uptake in the Hep G2 cell line, cells are exposed tobinding medium, washed, lysed, and assayed for radioactivity and totalprotein content in much the same way as described for TFR binding inExample 12. In this assay, however, radioactively-labeled LDL is usedand its uptake into cells after binding is studied by incubation at 37°C. During the incubation period, labeled LDL binds to LDLR and entersthe cell via the normal LDLR pathway. In addition, labeled LDL can alsobind to chimeric protein, and the LDLR/TF-LDL complex enters the cellafter binding to the TFR and uptake via the TFR pathway. Thus, unlabeledLDL inhibits binding of labeled LDL to both LDLR and chimeric protein,while human holo TF only inhibits binding of LDLR/TF-LDL complex to theTFR.

Radiolabeled (¹²⁵ I) human LDL (Biomedical Technologies, Inc.; BT-913R)at a specific activity of greater than 200 cpm per ng of LDL protein isdiluted in medium containing 80% DMEM, 0.5% protease-free bovinealbumin, and 20% PBS. Inhibitors of radiolabeled LDL binding and uptakeare diluted in PBS and added to this medium by substituting for the PBScomponent. In this example, uptake of ¹²⁵ I-LDL is measured in thepresence or absence of chimeric protein and in the presence or absenceof unlabeled inhibitors holo human TF (Sigma T-3400) or LDL (BiomedicalTechnologies, Inc. BT-903).

Hep G2 cells are plated into wells of 6-well tissue culture plates (35mm diameter) at 2×10⁵ cells per well and grown for 3 days in mediumconsisting of DMEM (Cellgro 50-013), 10% fetal bovine serum (HycloneA-1111), and 50 units/ml each of penicillin and streptomycin (GibcoBRL15070-014). Cells are then washed one to two times with DMEM andincubated at 37° C. with 5 ml of DMEM for 30 minutes. This washing andincubation step helps remove serum components introduced with the fetalbovine serum, including transferrin, that might otherwise be bound toTFR and interfere with the binding assay. After the 30 min. incubation,the medium in each well is removed by aspiration and replaced with oneml of uptake medium. The plates are placed at 37° C. in a standardtissue-culture incubator to allow uptake and accumulation of the labeledLDL.

After the uptake period, the plates are placed on ice and kept on iceduring subsequent washing steps. To remove unbound label from the cells,each well is washed 3 times with PBS containing 0.5% BSA, followed by anadditional 3 washes in PBS. The cells in each well are then lysed with 1ml of 1N NaOH and the lysates transferred to 12×75 mm polystyrene tubes(Falcon 2052). The wells are washed with an additional 1 ml of 1N NaOHwhich is added to the tube containing the appropriate lysate. The amountof radiolabeled LDL present in the lysates is determined byquantification of gamma emission in a gamma counter (Beckman). Becausethe cells internalize labeled LDL, the emissions counted reflect theamounts of labeled LDL or its metabolites inside the cells, as well asthe amounts of intact labeled LDL on the surface of the cells at the endof the uptake period. Lysates are also neutralized and assayed for totalprotein content by BCA Protein Assay (Pierce 23225G). The ratio of totalradioactivity (cpm) to total protein content (mg) gives a good estimateof LDL uptake. This assay can be used to show that the LDLR/TF chimericprotein promotes enhanced uptake of radiolabeled LDL into Hep G2 cellsand that unlabeled TF or LDL acts as a competitive inhibitor of thisenhanced uptake.

The uptake of LDL into cells can also be monitored by measuring theeffects of internalized LDL on intracellular processes and geneexpression, using established protocols (Goldstein, J. L. et al., Meth.Enzymol., 98:241-260 (1983)). Release of free cholesterol from LDLdirectly or by hydrolysis of cholesteryl esters results in a regulatorypool of cholesterol that not only suppresses the expression the geneencoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase),but also activates expression of the gene encoding acyl-CoA:cholesterolO-acyltransferase (ACAT). Since HMG-CoA reductase catalyzes therate-limiting step in cholesterol biosynthesis, down regulation of thegene encoding this enzyme reduces the de novo biosynthesis ofcholesterol as the levels of intracellular cholesterol increase due touptake stimulated by LDLR/TF. In addition, increased expression of ACAT,an enzyme that esterifies excess free cholesterol to allow storage inthe cytoplasm as cholesteryl ester droplets (principally cholesteryloleate), results in an increased capacity for storage of cholesterol bycells. Finally, synthesis of LDLRs is down regulated by an increase inthe regulatory pool of free cholesterol. Thus, these three secondaryeffects of increasing intracellular pools of cholesterol on cholesterolmetabolism constitute a mechanism of cholesterol homeostasis whichprotects the organism against overproduction of cholesterol.

When LDL-uptake occurs as a result of the chimeric protein functioningin the TFR-mediated endocytosis pathway, the free cholesterol releaseddoes not down-regulate the synthesis of TFRs as it does the synthesis ofLDLRs. The effect of this down-regulation would lead ultimately todecreased intracellular cholesterol (because of decreased uptake due tolower numbers of LDLRs) which subsequently leads to increasedcholesterol biosynthesis due increased activity of HMG-CoA reductase anddecreased ability to store cholesterol due to decreased ACAT activity.Continual uptake of LDL can occur in the presence of chimeric proteinvia the TFR-mediated endocytosis pathway, resulting in continuoussuppression of HMG-CoA reductase and continuous activation of ACAT.Thus, in bypassing the normal LDL uptake pathway, the decrease andincrease in biosynthesis and storage, respectively, of intracellularcholesterol can be maintained over long periods leading to sustainedreduced total serum cholesterol levels.

EXAMPLE 14 Effect of LDLR/TF Chimeric Protein on In Vivo MouseCholesterol Levels

To determine whether the LDLR/TF chimeric protein has ananti-hypercholesterolemic effect in an in vivo model system, anintravenous injection of purified chimeric protein is performed andcholesterol levels are measured at one or more time intervalspost-injection. Because an exogenously administered protein such asLDLR/TF will have a finite circulating lifetime in an animal, a bolusinjection should have only a transient effect in reducing levels of LDLcholesterol, with levels returning to baseline once the injected proteinis removed from the circulation. However, continuous delivery ofchimeric protein by means of a slow-release device or by gene therapy(implantation of cells genetically engineered to secrete chimericprotein) can have a long-term or permanent effect on LDL cholesterol.

Ishibashi et al. (J. Clin. Invest. 92:883-893 (1993)) describe anLDLR-knockout mouse that has increased levels of both total cholesteroland LDL cholesterol as compared to most strains of experimental mice.This mouse strain, which contains a homozygous deletion of the LDLRgene, is used for in vivo studies of the biological effects of LDLR/TFin a mammalian species.

In normal mice, most circulating cholesterol is carried by HDL, while inhumans most of the cholesterol is carried by LDL. Unlike humans,however, 70% of the LDL fraction in mice is associated with apoB-48, atruncated variant of apolipoprotein B lacking binding sites for LDLR,while 30% is associated with apoB-100, which contains the LDLR bindingregion. By contrast, 98% of human LDL contains apoB-100. In terms ofweight percentages, mouse LDL contains 9.5% unesterified cholesterol,23.5% cholesteryl esters, and 20.5% protein; human LDL contains 9.2%unesterified cholesterol, 37% cholesteryl esters, and 22% protein(Chapman, M., J. Meth. Enzymol. 128:70-143 (1986).

The effect of a homozygous deletion of the LDLR gene is to abolish theclearance of LDL (apoB-100), resulting in a higher steady-state level ofLDL. Homozygous female LDLR-knockout mice have total cholesterol levelsthat are 139 mg/dl higher than in the normal homozygous wild-typecontrols (Ishibashi et al. (J. Clin. Invest. 92:883-893 (1993)). Sincefunctional LDLR interacts with the apoB-100 LDL fraction but not theapoB-48 LDL fraction, and assuming that the excess 139 mg/dl of totalcholesterol is contained exclusively in the ApoB-100 LDL fraction, theapproximately 1 ml of serum in each mouse contains at least 1.39 mg oftotal cholesterol in the apoB-100 LDL fraction, the target molecule forLDLR/TF binding. Assuming that cholesterol comprises 33% of LDL byweight, that this fraction is 20.5% protein, and that the apoB-100molecule has a molecular weight of 514 kd, then 1.39 mg of totalcholesterol corresponds to approximately 1.68 nmol of apoB-100 per mouse(one apoB-100 molecule per LDL particle). For unimolecular binding toLDLR/TF (which has a molecular weight of 116.3 kd excludingcarbohydrate), 1.68 nmol of LDLR/TF corresponds to 195 μg of LDLR/TF permouse. To deliver LDLR/TF in an amount equal to, for example, 10% of thenumber of LDL (apoB-100) particles circulating in the LDLR-knockoutmouse (0.168 nmol) requires injection of 19.5 μg of LDLR/TF fusionprotein per mouse.

To establish baseline levels of total cholesterol, HDL cholesterol, andLDL cholesterol, retro-orbital bleeds are performed on anesthetizedanimals at certain times prior to injection with chimeric protein ornegative controls. In each animal, serum is isolated from clotted bloodand tested for levels of total cholesterol and HDL cholesterol, using acoupled enzymatic assay with colorimetric endpoint (Sigma Chemical Co.,St. Louis, Mo.). Total cholesterol concentration in mg/dl is quantifiedby adapting the manual procedure to a 96-well microtiter plate format.Sera and cholesterol standards are diluted in normal saline solution and10 μl of each are loaded into duplicate or triplicate wells of a 96-wellmicrotiter plate. 200 μl of Cholesterol Reagent (Sigma 352-20) are addedto each well and the plate is incubated at 37° C. for 10 minutes, afterwhich the absorbance at 490 nm is read on a microplate reader. In thisassay, absorbance is linear between 0 and 200 mg/dl (standard curvegenerated with cholesterol standards; Sigma C-0534) and samples may bediluted to fall within this range.

HDL cholesterol concentration in mg/dl is quantified as totalcholesterol (performed as above) following precipitation of LDL and VLDLcholesterol fractions with HDL Cholesterol Reagent (Sigma 352-3). Serumis mixed with one-tenth volume of HDL Cholesterol Reagent and allowed tostand at room temperature for approximately 5 minutes. Followingcentrifugation in a microcentrifuge for 2 minutes at high speed, aportion of the supernatant is removed, diluted in normal saline, andassayed for total cholesterol by the above method. To obtain the HDLcholesterol concentration in the original serum, the total cholesterolconcentration in the assayed supernatant is multiplied by 1.1 to accountfor the 10% increase in volume due to addition of HDL CholesterolReagent. LDL cholesterol is calculated by subtracting the value obtainedfor HDL cholesterol from the total cholesterol value obtained prior totreatment of serum with the HDL Cholesterol Reagent and removal of theLDL and VLDL fractions.

After pre-injection baseline levels of serum cholesterol have beendetermined, 50 μl each of an appropriate amount of LDLR/TF chimericprotein (in phosphate-buffered saline; PBS) is injected into the tailveins of anesthetized LDLR-knockout mice. To assess the effects ofanesthesia and injection, 50 μl of PBS is injected into differentLDLR-knockout animals as negative controls. To determine if interactionof the TF receptor with administered TF domains affects lipoproteinmetabolism, 50 μl of a PBS solution containing human holotransferrin(holo TF) at the same molar concentration as LDLR/TF is injected intoLDLR-knockout animals as additional negative controls. Human holo TFcontains the same TF domain as contained in the LDLR/TF chimericprotein, but lacks the LDLR domain and cannot bind LDL. This type ofnegative control permits evaluation of any effects that may result frominjection of an equivalent amount of a human protein or human TFantigen.

Following injections of chimeric protein or negative-control substances,retro-orbital bleeds are performed again at time intervals appropriatefor the given volume and frequency of sampling. Sera from post-injectionbleeds are assayed for the concentrations of total cholesterol and HDLcholesterol. Levels of LDL (i.e. LDL plus VLDL) cholesterol arecalculated as the arithmetic difference between total cholesterol andHDL cholesterol concentrations. In this manner, the effect that LDLR/TFhas on decreasing specific serum cholesterol fractions may bequantified.

Sera from post-injection bleeds can also be assayed for theconcentration of LDLR/TF chimeric protein remaining in the circulation.The human TF ELISA (Example 6) can be used, since the antibodiesemployed in the assay do not cross-react with mouse transferrin.Measurement of the rate of disappearance of chimeric protein in mousesera allows an estimation of the circulating half-life of chimericprotein.

The effects of specific chimeric proteins on lipoprotein clearance ratescan also be studied. Ishibashi et al. (J. Clin. Invest. 92:883-893(1993) have shown that loss of the LDLR in mice reduces significantlythe rate of clearance of injected radiolabeled LDL or VLDL, but does notalter the clearance rate of injected radiolabeled HDL. Clearance ratesfollowing chimeric protein delivery (bolus or continuous infusion) canbe studied in this way in order to quantify the increase in lipoproteinclearance rate resulting from administration of the chimeric protein.

In addition to the LDLR knockout mouse, the Watanabe rabbit, which hasdefective LDL receptors and hypercholesterolemia due to defective LDLmetabolism, may be used to assess the in vivo effects of the LDLR/TFchimeric protein on cholesterol levels.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3427 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGAGGCTGCGAGCATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCT60                CGCCGCGGCGGGGACTGCAGTGGGCGACAGATGTGAAAGAAACGAGTTCCAGTGCCAAGA120               CGGGAAATGCATCTCCTACAAGTGGGTCTGCGATGGCAGCGCTGAGTGCCAGGATGGCTC180               TGATGAGTCCCAGGAGACGTGCTTGTCTGTCACCTGCAAATCCGGGGACTTCAGCTGTGG240               GGGCCGTGTCAACCGCTGCATTCCTCAGTTCTGGAGGTGCGATGGCCAAGTGGACTGCGA300               CAACGGCTCAGACGAGCAAGGCTGTCCCCCCAAGACGTGCTCCCAGGACGAGTTTCGCTG360               CCACGATGGGAAGTGCATCTCTCGGCAGTTCGTCTGTGACTCAGACCGGGACTGCTTGGA420               CGGCTCAGACGAGGCCTCCTGCCCGGTGCTCACCTGTGGTCCCGCCAGCTTCCAGTGCAA480               CAGCTCCACCTGCATCCCCCAGCTGTGGGCCTGCGACAACGACCCCGACTGCGAAGATGG540               CTCGGATGAGTGGCCGCAGCGCTGTAGGGGTCTTTACGTGTTCCAAGGGGACAGTAGCCC600               CTGCTCGGCCTTCGAGTTCCACTGCCTAAGTGGCGAGTGCATCCACTCCAGCTGGCGCTG660               TGATGGTGGCCCCGACTGCAAGGACAAATCTGACGAGGAAAACTGCGCTGTGGCCACCTG720               TCGCCCTGACGAATTCCAGTGCTCTGATGGAAACTGCATCCATGGCAGCCGGCAGTGTGA780               CCGGGAATATGACTGCAAGGACATGAGCGATGAAGTTGGCTGCGTTAATGTGACACTCTG840               CGAGGGACCCAACAAGTTCAAGTGTCACAGCGGCGAATGCATCACCCTGGACAAAGTCTG900               CAACATGGCTAGAGACTGCCGGGACTGGTCAGATGAACCCATCAAAGAGTGCGGGACCAA960               CGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTA1020              CGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATATCGA1080              TGAGTGTCAGGATCCCGACACCTGCAGCCAGCTCTGCGTGAACCTGGAGGGTGGCTACAA1140              GTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAGGCTGTGGT1200              CCCTGATAAAACTGTGAGATGGTGTGCAGTGTCGGAGCATGAGGCCACTAAGTGCCAGAG1260              TTTCCGCGACCATATGAAAAGCGTCATTCCATCCGATGGTCCCAGTGTTGCTTGTGTGAA1320              GAAAGCCTCCTACCTTGATTGCATCAGGGCCATTGCGGCAAACGAAGCGGATGCTGTGAC1380              ACTGGATGCAGGTTTGGTGTATGATGCTTACTTGGCTCCCAATAACCTGAAGCCTGTGGT1440              GGCAGAGTTCTATGGGTCAAAAGAGGATCCACAGACTTTCTATTATGCTGTTGCTGTGGT1500              GAAGAAGGATAGTGGCTTCCAGATGAACCAGCTTCGAGGCAAGAAGTCCTGCCACACGGG1560              TCTAGGCAGGTCCGCTGGGTGGAACATCCCCATAGGCTTACTTTACTGTGACTTACCTGA1620              GCCACGTAAACCTCTTGAGAAAGCAGTGGCCAATTTCTTCTCGGGCAGCTGTGCCCCTTG1680              TGCGGATGGGACGGACTTCCCCCAGCTGTGTCAACTGTGTCCAGGGTGTGGCTGCTCCAC1740              CCTTAACCAATACTTCGGCTACTCGGGAGCCTTCAAGTGTCTGAAGGATGGTGCTGGGGA1800              TGTGGCCTTTGTCAAGCACTCGACTATATTTGAGAACTTGGCAAACAAGGCTGACAGGGA1860              CCAGTATGAGCTGCTTTGCCTAGACAACACCCGGAAGCCGGTAGATGAATACAAGGACTG1920              CCACTTGGCCCAGGTCCCTTCTCATACCGTCGTGGCCCGAAGTATGGGCGGCAAGGAGGA1980              CTTGATCTGGGAGCTTCTCAACCAGGCCCAGGAACATTTTGGCAAAGACAAATCAAAAGA2040              ATTCCAACTATTCAGCTCTCCCATGGGAAGGACCTGCTGTTTAAGGACTCTGCCCACGGG2100              TTTTTAAAAGTCCCCCCAAGGATGGATGCCAAGATGTACCTGGGCTATGAGTATGTCACT2160              GCCATCCGGAATCTACGGGAAGGCACATGCCCAGAAGCCCCAACAGATGAATGCAAGCCT2220              GTGAAGTGGTGTGCGCTGAGCCACCACGAGAGGCTCAAGTGTGATGAGTGGAGTGTTAAC2280              AGTGTAGGGAAAATAGAGTGTGTATCAGCAGAGACCACCGAAGACTGCATCGCCAAGATC2340              ATGAATGGAGAAGCTGATGCCATGAGCTTGGATGGAGGGTTTGTCTACATAGCGGGCAAG2400              TGTGGTCTGGTGCCTGTCTTGGCAGAAAACTACAATAAGAGCGATAATTGTGAGGATACA2460              CCAGAGGCAGGGTATTTTGCTGTAGCAGTGGTGAAGAAATCAGCTTCTGACCTCACCTGG2520              GACAATCTGAAAGGCAAGAAGTCCTGCCATACGGCAGTTGGCAGAACCGCTGGCTGGAAC2580              ATCCCCATGGGCCTGCTCTACAATAAGATCAACCACTGCAGATTTGATGAATTTTTCAGT2640              GAAGGTTGTGCCCCTGGGTCTAAGAAAGACTCCAGTCTCTGTAAGCTGTGTATGGGCTCA2700              GGCCTAAACCTGTGTGAACCCAACAACAAAGAGGGATACTACGGCTACACAGGCGCTTTC2760              AGGTGTCTGGTTGAGAAGGGAGATGTGGCCTTTGTGAAACACCAGACTGTCCCACAGAAC2820              ACTGGGGGAAAAAACCCTGATCCATGGGCTAAGAATCTGAATGAAAAAGACTATGAGTTG2880              CTGTGCCTTGATGGTACCAGGAAACCTGTGGAGGAGTATGCGAACTGCCACCTGGCCAGA2940              GCCCCGAATCACGCTGTGGTCACACGGAAAGATAAGGAAGCTTGCGTCCACAAGATATTA3000              CGTCAACAGCAGCACCTATTTGGAAGCAACGTAACTGACTGCTCGGGCAACTTTTGTTTG3060              TTCCGGTCGGAAACCAAGGACCTTCTGTTCAGAGATGACACAGTATGTTTGGCCAAACTT3120              CATGACAGAAACACATATGAAAAATACTTAGGAGAAGAATATGTCAAGGCTGTTGGTAAC3180              CTGAGAAAATGCTCCACCTCATCACTCCTGGAAGCCTGCACTTTCCGTAGACCTTAAAAT3240              CTCAGAGGTAGGGCTGCCACCAAGGTGAAGATGGGAACGCAGATGATCCATGAGTTTGCC3300              CTGGTTTCACTGGCCCAAGTGGTTTGTGCTAACCACGTCTGTCTTCACAGCTCTGTGTTG3360              CCATGTGTGCTGAACAAAAAATAAAAATTATTATTGATTTTATATTTCGGGGGGGGGGCT3420              GCAGCCC3427                                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1074 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyProTrpGlyTrpLysLeuArgTrpThrValAlaLeuLeuLeu                              151015                                                                        AlaAlaAlaGlyThrAlaValGlyAspArgCysGluArgAsnGluPhe                              202530                                                                        GlnCysGlnAspGlyLysCysIleSerTyrLysTrpValCysAspGly                              354045                                                                        SerAlaGluCysGlnAspGlySerAspGluSerGlnGluThrCysLeu                              505560                                                                        SerValThrCysLysSerGlyAspPheSerCysGlyGlyArgValAsn                              65707580                                                                      ArgCysIleProGlnPheTrpArgCysAspGlyGlnValAspCysAsp                              859095                                                                        AsnGlySerAspGluGlnGlyCysProProLysThrCysSerGlnAsp                              100105110                                                                     GluPheArgCysHisAspGlyLysCysIleSerArgGlnPheValCys                              115120125                                                                     AspSerAspArgAspCysLeuAspGlySerAspGluAlaSerCysPro                              130135140                                                                     ValLeuThrCysGlyProAlaSerPheGlnCysAsnSerSerThrCys                              145150155160                                                                  IleProGlnLeuTrpAlaCysAspAsnAspProAspCysGluAspGly                              165170175                                                                     SerAspGluTrpProGlnArgCysArgGlyLeuTyrValPheGlnGly                              180185190                                                                     AspSerSerProCysSerAlaPheGluPheHisCysLeuSerGlyGlu                              195200205                                                                     CysIleHisSerSerTrpArgCysAspGlyGlyProAspCysLysAsp                              210215220                                                                     LysSerAspGluGluAsnCysAlaValAlaThrCysArgProAspGlu                              225230235240                                                                  PheGlnCysSerAspGlyAsnCysIleHisGlySerArgGlnCysAsp                              245250255                                                                     ArgGluTyrAspCysLysAspMetSerAspGluValGlyCysValAsn                              260265270                                                                     ValThrLeuCysGluGlyProAsnLysPheLysCysHisSerGlyGlu                              275280285                                                                     CysIleThrLeuAspLysValCysAsnMetAlaArgAspCysArgAsp                              290295300                                                                     TrpSerAspGluProIleLysGluCysGlyThrAsnGluCysLeuAsp                              305310315320                                                                  AsnAsnGlyGlyCysSerHisValCysAsnAspLeuLysIleGlyTyr                              325330335                                                                     GluCysLeuCysProAspGlyPheGlnLeuValAlaGlnArgArgCys                              340345350                                                                     GluAspIleAspGluCysGlnAspProAspThrCysSerGlnLeuCys                              355360365                                                                     ValAsnLeuGluGlyGlyTyrLysCysGlnCysGluGluGlyPheGln                              370375380                                                                     LeuAspProHisThrLysAlaCysLysAlaValValProAspLysThr                              385390395400                                                                  ValArgTrpCysAlaValSerGluHisGluAlaThrLysCysGlnSer                              405410415                                                                     PheArgAspHisMetLysSerValIleProSerAspGlyProSerVal                              420425430                                                                     AlaCysValLysLysAlaSerTyrLeuAspCysIleArgAlaIleAla                              435440445                                                                     AlaAsnGluAlaAspAlaValThrLeuAspAlaGlyLeuValTyrAsp                              450455460                                                                     AlaTyrLeuAlaProAsnAsnLeuLysProValValAlaGluPheTyr                              465470475480                                                                  GlySerLysGluAspProGlnThrPheTyrTyrAlaValAlaValVal                              485490495                                                                     LysLysAspSerGlyPheGlnMetAsnGlnLeuArgGlyLysLysSer                              500505510                                                                     CysHisThrGlyLeuGlyArgSerAlaGlyTrpAsnIleProIleGly                              515520525                                                                     LeuLeuTyrCysAspLeuProGluProArgLysProLeuGluLysAla                              530535540                                                                     ValAlaAsnPhePheSerGlySerCysAlaProCysAlaAspGlyThr                              545550555560                                                                  AspPheProGlnLeuCysGlnLeuCysProGlyCysGlyCysSerThr                              565570575                                                                     LeuAsnGlnTyrPheGlyTyrSerGlyAlaPheLysCysLeuLysAsp                              580585590                                                                     GlyAlaGlyAspValAlaPheValLysHisSerThrIlePheGluAsn                              595600605                                                                     LeuAlaAsnLysAlaAspArgAspGlnTyrGluLeuLeuCysLeuAsp                              610615620                                                                     AsnThrArgLysProValAspGluTyrLysAspCysHisLeuAlaGln                              625630635640                                                                  ValProSerHisThrValValAlaArgSerMetGlyGlyLysGluAsp                              645650655                                                                     LeuIleTrpGluLeuLeuAsnGlnAlaGlnGluHisPheGlyLysAsp                              660665670                                                                     LysSerLysGluPheGlnLeuPheSerSerProHisGlyLysAspLeu                              675680685                                                                     LeuPheLysAspSerAlaHisGlyPheLeuLysValProProArgMet                              690695700                                                                     AspAlaLysMetTyrLeuGlyTyrGluTyrValThrAlaIleArgAsn                              705710715720                                                                  LeuArgGluGlyThrCysProGluAlaProThrAspGluCysLysPro                              725730735                                                                     ValLysTrpCysAlaLeuSerHisHisGluArgLeuLysCysAspGlu                              740745750                                                                     TrpSerValAsnSerValGlyLysIleGluCysValSerAlaGluThr                              755760765                                                                     ThrGluAspCysIleAlaLysIleMetAsnGlyGluAlaAspAlaMet                              770775780                                                                     SerLeuAspGlyGlyPheValTyrIleAlaGlyLysCysGlyLeuVal                              785790795800                                                                  ProValLeuAlaGluAsnTyrAsnLysSerAspAsnCysGluAspThr                              805810815                                                                     ProGluAlaGlyTyrPheAlaValAlaValValLysLysSerAlaSer                              820825830                                                                     AspLeuThrTrpAspAsnLeuLysGlyLysLysSerCysHisThrAla                              835840845                                                                     ValGlyArgThrAlaGlyTrpAsnIleProMetGlyLeuLeuTyrAsn                              850855860                                                                     LysIleAsnHisCysArgPheAspGluPhePheSerGluGlyCysAla                              865870875880                                                                  ProGlySerLysLysAspSerSerLeuCysLysLeuCysMetGlySer                              885890895                                                                     GlyLeuAsnLeuCysGluProAsnAsnLysGluGlyTyrTyrGlyTyr                              900905910                                                                     ThrGlyAlaPheArgCysLeuValGluLysGlyAspValAlaPheVal                              915920925                                                                     LysHisGlnThrValProGlnAsnThrGlyGlyLysAsnProAspPro                              930935940                                                                     TrpAlaLysAsnLeuAsnGluLysAspTyrGluLeuLeuCysLeuAsp                              945950955960                                                                  GlyThrArgLysProValGluGluTyrAlaAsnCysHisLeuAlaArg                              965970975                                                                     AlaProAsnHisAlaValValThrArgLysAspLysGluAlaCysVal                              980985990                                                                     HisLysIleLeuArgGlnGlnGlnHisLeuPheGlySerAsnValThr                              99510001005                                                                   AspCysSerGlyAsnPheCysLeuPheArgSerGluThrLysAspLeu                              101010151020                                                                  LeuPheArgAspAspThrValCysLeuAlaLysLeuHisAspArgAsn                              1025103010351040                                                              ThrTyrGluLysTyrLeuGlyGluGluTyrValLysAlaValGlyAsn                              104510501055                                                                  LeuArgLysCysSerThrSerSerLeuLeuGluAlaCysThrPheArg                              106010651070                                                                  ArgPro                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4601 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGAGGCTGCGAGCATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCT60                CGCCGCGGCGGGGACTGCAGTGGGCGACAGATGTGAAAGAAACGAGTTCCAGTGCCAAGA120               CGGGAAATGCATCTCCTACAAGTGGGTCTGCGATGGCAGCGCTGAGTGCCAGGATGGCTC180               TGATGAGTCCCAGGAGACGTGCTTGTCTGTCACCTGCAAATCCGGGGACTTCAGCTGTGG240               GGGCCGTGTCAACCGCTGCATTCCTCAGTTCTGGAGGTGCGATGGCCAAGTGGACTGCGA300               CAACGGCTCAGACGAGCAAGGCTGTCCCCCCAAGACGTGCTCCCAGGACGAGTTTCGCTG360               CCACGATGGGAAGTGCATCTCTCGGCAGTTCGTCTGTGACTCAGACCGGGACTGCTTGGA420               CGGCTCAGACGAGGCCTCCTGCCCGGTGCTCACCTGTGGTCCCGCCAGCTTCCAGTGCAA480               CAGCTCCACCTGCATCCCCCAGCTGTGGGCCTGCGACAACGACCCCGACTGCGAAGATGG540               CTCGGATGAGTGGCCGCAGCGCTGTAGGGGTCTTTACGTGTTCCAAGGGGACAGTAGCCC600               CTGCTCGGCCTTCGAGTTCCACTGCCTAAGTGGCGAGTGCATCCACTCCAGCTGGCGCTG660               TGATGGTGGCCCCGACTGCAAGGACAAATCTGACGAGGAAAACTGCGCTGTGGCCACCTG720               TCGCCCTGACGAATTCCAGTGCTCTGATGGAAACTGCATCCATGGCAGCCGGCAGTGTGA780               CCGGGAATATGACTGCAAGGACATGAGCGATGAAGTTGGCTGCGTTAATGTGACACTCTG840               CGAGGGACCCAACAAGTTCAAGTGTCACAGCGGCGAATGCATCACCCTGGACAAAGTCTG900               CAACATGGCTAGAGACTGCCGGGACTGGTCAGATGAACCCATCAAAGAGTGCGGGACCAA960               CGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTA1020              CGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATATCGA1080              TGAGTGTCAGGATCCCGACACCTGCAGCCAGCTCTGCGTGAACCTGGAGGGTGGCTACAA1140              GTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAGGCTGTGGG1200              CTCCATCGCCTACCTCTTCTTCACCAACCGGCACGAGGTCAGGAAGATGACGCTGGACCG1260              GAGCGAGTACACCAGCCTCATCCCCAACCTGAGGAACGTGGTCGCTCTGGACACGGAGGT1320              GGCCAGCAATAGAATCTACTGGTCTGACCTGTCCCAGAGAATGATCTGCAGCACCCAGCT1380              TGACAGAGCCCACGGCGTCTCTTCCTATGACACCGTCATCAGCAGGGACATCCAGGCCCC1440              CGACGGGCTGGCTGTGGACTGGATCCACAGCAACATCTACTGGACCGACTCTGTCCTGGG1500              CACTGTCTCTGTTGCGGATACCAAGGGCGTGAAGAGGAAAACGTTATTCAGGGAGAACGG1560              CTCCAAGCCAAGGGCCATCGTGGTGGATCCTGTTCATGGCTTCATGTACTGGACTGACTG1620              GGGAACTCCCGCCAAGATCAAGAAAGGGGGCCTGAATGGTGTGGACATCTACTCGCTGGT1680              GACTGAAAACATTCAGTGGCCCAATGGCATCACCCTAGATCTCCTCAGTGGCCGCCTCTA1740              CTGGGTTGACTCCAAACTTCACTCCATCTCAAGCATCGATGTCAATGGGGGCAACCGGAA1800              GACCATCTTGGAGGATGAAAAGAGGCTGGCCCACCCCTTCTCCTTGGCCGTCTTTGAGGA1860              CAAAGTATTTTGGACAGATATCATCAACGAAGCCATTTTCAGTGCCAACCGCCTCACAGG1920              TTCCGATGTCAACTTGTTGGCTGAAAACCTACTGTCCCCAGAGGATATGGTCCTCTTCCA1980              CAACCTCACCCAGCCAAGAGGAGTGAACTGGTGTGAGAGGACCACCCTGAGCAATGGCGG2040              CTGCCAGTATCTGTGCCTCCCGCCCCGCAGATCAACCCCCACTCGCCCAAGTTTACCTGC2100              GCCTGCCCGGACGGCATGCTGCTGGCCAGGGACATGAGGAGCTGCCTCACAGAGGCTGAG2160              GCTGCAGTGGCCACCCAGGAGACATCCACCGTCAGGCTAAAGGTCGTCCCTGATAAAACT2220              GTGAGATGGTGTGCAGTGTCGGAGCATGAGGCCACTAAGTGCCAGAGTTTCCGCGACCAT2280              ATGAAAAGCGTCATTCCATCCGATGGTCCCAGTGTTGCTTGTGTGAAGAAAGCCTCCTAC2340              CTTGATTGCATCAGGGCCATTGCGGCAAACGAAGCGGATGCTGTGACACTGGATGCAGGT2400              TTGGTGTATGATGCTTACTTGGCTCCCAATAACCTGAAGCCTGTGGTGGCAGAGTTCTAT2460              GGGTCAAAAGAGGATCCACAGACTTTCTATTATGCTGTTGCTGTGGTGAAGAAGGATAGT2520              GGCTTCCAGATGAACCAGCTTCGAGGCAAGAAGTCCTGCCACACGGGTCTAGGCAGGTCC2580              GCTGGGTGGAACATCCCCATAGGCTTACTTTACTGTGACTTACCTGAGCCACGTAAACCT2640              CTTGAGAAAGCAGTGGCCAATTTCTTCTCGGGCAGCTGTGCCCCTTGTGCGGATGGGACG2700              GACTTCCCCCAGCTGTGTCAACTGTGTCCAGGGTGTGGCTGCTCCACCCTTAACCAATAC2760              TTCGGCTACTCGGGAGCCTTCAAGTGTCTGAAGGATGGTGCTGGGGATGTGGCCTTTGTC2820              AAGCACTCGACTATATTTGAGAACTTGGCAAACAAGGCTGACAGGGACCAGTATGAGCTG2880              CTTTGCCTAGACAACACCCGGAAGCCGGTAGATGAATACAAGGACTGCCACTTGGCCCAG2940              GTCCCTTCTCATACCGTCGTGGCCCGAAGTATGGGCGGCAAGGAGGACTTGATCTGGGAG3000              CTTCTCAACCAGGCCCAGGAACATTTTGGCAAAGACAAATCAAAAGAATTCCAACTATTC3060              AGCTCTCCTCATGGGAAGGACCTGCTGTTTAAGGACTCTGCCCACGGGTTTTTAAAAGTC3120              CCCCCAAGGATGGATGCCAAGATGTACCTGGGCTATGAGTATGTCACTGCCATCCGGAAT3180              CTACGGGAAGGCACATGCCCAGAAGCCCCAACAGATGAATGCAAGCCTGTGAAGTGGTGT3240              GCGCTGAGCCACCACGAGAGGCTCAAGTGTGATGAGTGGAGTGTTAACAGTGTAGGGAAA3300              ATAGAGTGTGTATCAGCAGAGACCACCGAAGACTGCATCGCCAAGATCATGAATGGAGAA3360              GCTGATGCCATGAGCTTGGATGGAGGGTTTGTCTACATAGCGGGCAAGTGTGGTCTGGTG3420              CCTGTCTTGGCAGAAAACTACAATAAGAGCGATAATTGTGAGGATACACCAGAGGCAGGG3480              TATTTTGCTGTAGCAGTGGTGAAGAAATCAGCTTCTGACCTCACCTGGGACAATCTGAAA3540              GGCAAGAAGTCCTGCCATACGGCAGTTGGCAGAACCGCTGGCTGGAACATCCCCATGGGC3600              CTGCTCTACAATAAGATCAACCACTGCAGATTTGATGAATTTTTCAGTGAAGGTTGTGCC3660              CCTGGGTCTAAGAAAGACTCCAGTCTCTGTAAGCTGTGTATGGGCTCAGGCCTAAACCTG3720              TGTGAACCCAACAACAAAGAGGGATACTACGGCTACACAGGCGCTTTCAGGTGTCTGGTT3780              GAGAAGGGAGATGTGGCCTTTGTGAAACACCAGACTGTCCCACAGAACACTGGGGGAAAA3840              AACCCTGATCCATGGGCTAAGAATCTGAATGAAAAAGACTATGAGTTGCTGTGCCTTGAT3900              GGTACCAGGAAACCTGTGGAGGAGTATGCGAACTGCCACCTGGCCAGAGCCCCGAATCAC3960              GCTGTGGTCACACGGAAAGATAAGGAAGCTTGCGTCCACAAGATATTACGTCAACAGCAG4020              CACCTATTTGGAAGCAACGTAACTGACTGCTCGGGCAACTTTTGTTTGTTCCGGTCGGAA4080              ACCAAGGACCTTCTGTTCAGAGATGACACGTATGTTTGGCCAAACTTCATGACAGAAACA4140              CATATGAAAAATACTTAGGAGAAGAATATGTCAAGGCTGTTGGTAACCTGAGAAAATGCT4200              CCACCTCATCACTCCTGGAAGCCTGCACTTTCCGTAGACCTTAAAATCTCAGAGGTAGGG4260              CTGCCACCAAGGTGAAGATGGGAACGCAGATGATCCATGAGTTTGCCCTGGTTTCACTGG4320              CCCAAGTGGTTTGTGCTAACCACGTCTGTCTTCACAGCTCTGTGTTGCCATGTGTGCTGA4380              ACAAAAAATAAAAATTATTATTGATTTTATATTTCGGGGGGGGGGCTGCAGCCCCTAGAC4440              CTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTCTCCCACGTGGGAGACAAGAAA4500              TCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTGGGTCCTTGCACCCCTCACTCTG4560              GCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTCTAGA4601                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1410 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGlyProTrpGlyTrpLysLeuArgTrpThrValAlaLeuLeuLeu                              151015                                                                        AlaAlaAlaGlyThrAlaValGlyAspArgCysGluArgAsnGluPhe                              202530                                                                        GlnCysGlnAspGlyLysCysIleSerTyrLysTrpValCysAspGly                              354045                                                                        SerAlaGluCysGlnAspGlySerAspGluSerGlnGluThrCysLeu                              505560                                                                        SerValThrCysLysSerGlyAspPheSerCysGlyGlyArgValAsn                              65707580                                                                      ArgCysIleProGlnPheTrpArgCysAspGlyGlnValAspCysAsp                              859095                                                                        AsnGlySerAspGluGlnGlyCysProProLysThrCysSerGlnAsp                              100105110                                                                     GluPheArgCysHisAspGlyLysCysIleSerArgGlnPheValCys                              115120125                                                                     AspSerAspArgAspCysLeuAspGlySerAspGluAlaSerCysPro                              130135140                                                                     ValLeuThrCysGlyProAlaSerPheGlnCysAsnSerSerThrCys                              145150155160                                                                  IleProGlnLeuTrpAlaCysAspAsnAspProAspCysGluAspGly                              165170175                                                                     SerAspGluTrpProGlnArgCysArgGlyLeuTyrValPheGlnGly                              180185190                                                                     AspSerSerProCysSerAlaPheGluPheHisCysLeuSerGlyGlu                              195200205                                                                     CysIleHisSerSerTrpArgCysAspGlyGlyProAspCysLysAsp                              210215220                                                                     LysSerAspGluGluAsnCysAlaValAlaThrCysArgProAspGlu                              225230235240                                                                  PheGlnCysSerAspGlyAsnCysIleHisGlySerArgGlnCysAsp                              245250255                                                                     ArgGluTyrAspCysLysAspMetSerAspGluValGlyCysValAsn                              260265270                                                                     ValThrLeuCysGluGlyProAsnLysPheLysCysHisSerGlyGlu                              275280285                                                                     CysIleThrLeuAspLysValCysAsnMetAlaArgAspCysArgAsp                              290295300                                                                     TrpSerAspGluProIleLysGluCysGlyThrAsnGluCysLeuAsp                              305310315320                                                                  AsnAsnGlyGlyCysSerHisValCysAsnAspLeuLysIleGlyTyr                              325330335                                                                     GluCysLeuCysProAspGlyPheGlnLeuValAlaGlnArgArgCys                              340345350                                                                     GluAspIleAspGluCysGlnAspProAspThrCysSerGlnLeuCys                              355360365                                                                     ValAsnLeuGluGlyGlyTyrLysCysGlnCysGluGluGlyPheGln                              370375380                                                                     LeuAspProHisThrLysAlaCysLysAlaValGlySerIleAlaTyr                              385390395400                                                                  LeuPhePheThrAsnArgHisGluValArgLysMetThrLeuAspArg                              405410415                                                                     SerGluTyrThrSerLeuIleProAsnLeuArgAsnValValAlaLeu                              420425430                                                                     AspThrGluValAlaSerAsnArgIleTyrTrpSerAspLeuSerGln                              435440445                                                                     ArgMetIleCysSerThrGlnLeuAspArgAlaHisGlyValSerSer                              450455460                                                                     TyrAspThrValIleSerArgAspIleGlnAlaProAspGlyLeuAla                              465470475480                                                                  ValAspTrpIleHisSerAsnIleTyrTrpThrAspSerValLeuGly                              485490495                                                                     ThrValSerValAlaAspThrLysGlyValLysArgLysThrLeuPhe                              500505510                                                                     ArgGluAsnGlySerLysProArgAlaIleValValAspProValHis                              515520525                                                                     GlyPheMetTyrTrpThrAspTrpGlyThrProAlaLysIleLysLys                              530535540                                                                     GlyGlyLeuAsnGlyValAspIleTyrSerLeuValThrGluAsnIle                              545550555560                                                                  GlnTrpProAsnGlyIleThrLeuAspLeuLeuSerGlyArgLeuTyr                              565570575                                                                     TrpValAspSerLysLeuHisSerIleSerSerIleAspValAsnGly                              580585590                                                                     GlyAsnArgLysThrIleLeuGluAspGluLysArgLeuAlaHisPro                              595600605                                                                     PheSerLeuAlaValPheGluAspLysValPheTrpThrAspIleIle                              610615620                                                                     AsnGluAlaIlePheSerAlaAsnArgLeuThrGlySerAspValAsn                              625630635640                                                                  LeuLeuAlaGluAsnLeuLeuSerProGluAspMetValLeuPheHis                              645650655                                                                     AsnLeuThrGlnProArgGlyValAsnTrpCysGluArgThrThrLeu                              660665670                                                                     SerAsnGlyGlyCysGlnTyrLeuCysLeuProAlaProGlnIleAsn                              675680685                                                                     ProHisSerProLysPheThrCysAlaCysProAspGlyMetLeuLeu                              690695700                                                                     AlaArgAspMetArgSerCysLeuThrGluAlaGluAlaAlaValAla                              705710715720                                                                  ThrGlnGluThrSerThrValArgLeuLysValValProAspLysThr                              725730735                                                                     ValArgTrpCysAlaValSerGluHisGluAlaThrLysCysGlnSer                              740745750                                                                     PheArgAspHisMetLysSerValIleProSerAspGlyProSerVal                              755760765                                                                     AlaCysValLysLysAlaSerTyrLeuAspCysIleArgAlaIleAla                              770775780                                                                     AlaAsnGluAlaAspAlaValThrLeuAspAlaGlyLeuValTyrAsp                              785790795800                                                                  AlaTyrLeuAlaProAsnAsnLeuLysProValValAlaGluPheTyr                              805810815                                                                     GlySerLysGluAspProGlnThrPheTyrTyrAlaValAlaValVal                              820825830                                                                     LysLysAspSerGlyPheGlnMetAsnGlnLeuArgGlyLysLysSer                              835840845                                                                     CysHisThrGlyLeuGlyArgSerAlaGlyTrpAsnIleProIleGly                              850855860                                                                     LeuLeuTyrCysAspLeuProGluProArgLysProLeuGluLysAla                              865870875880                                                                  ValAlaAsnPhePheSerGlySerCysAlaProCysAlaAspGlyThr                              885890895                                                                     AspPheProGlnLeuCysGlnLeuCysProGlyCysGlyCysSerThr                              900905910                                                                     LeuAsnGlnTyrPheGlyTyrSerGlyAlaPheLysCysLeuLysAsp                              915920925                                                                     GlyAlaGlyAspValAlaPheValLysHisSerThrIlePheGluAsn                              930935940                                                                     LeuAlaAsnLysAlaAspArgAspGlnTyrGluLeuLeuCysLeuAsp                              945950955960                                                                  AsnThrArgLysProValAspGluTyrLysAspCysHisLeuAlaGln                              965970975                                                                     ValProSerHisThrValValAlaArgSerMetGlyGlyLysGluAsp                              980985990                                                                     LeuIleTrpGluLeuLeuAsnGlnAlaGlnGluHisPheGlyLysAsp                              99510001005                                                                   LysSerLysGluPheGlnLeuPheSerSerProHisGlyLysAspLeu                              101010151020                                                                  LeuPheLysAspSerAlaHisGlyPheLeuLysValProProArgMet                              1025103010351040                                                              AspAlaLysMetTyrLeuGlyTyrGluTyrValThrAlaIleArgAsn                              104510501055                                                                  LeuArgGluGlyThrCysProGluAlaProThrAspGluCysLysPro                              106010651070                                                                  ValLysTrpCysAlaLeuSerHisHisGluArgLeuLysCysAspGlu                              107510801085                                                                  TrpSerValAsnSerValGlyLysIleGluCysValSerAlaGluThr                              109010951100                                                                  ThrGluAspCysIleAlaLysIleMetAsnGlyGluAlaAspAlaMet                              1105111011151120                                                              SerLeuAspGlyGlyPheValTyrIleAlaGlyLysCysGlyLeuVal                              112511301135                                                                  ProValLeuAlaGluAsnTyrAsnLysSerAspAsnCysGluAspThr                              114011451150                                                                  ProGluAlaGlyTyrPheAlaValAlaValValLysLysSerAlaSer                              115511601165                                                                  AspLeuThrTrpAspAsnLeuLysGlyLysLysSerCysHisThrAla                              117011751180                                                                  ValGlyArgThrAlaGlyTrpAsnIleProMetGlyLeuLeuTyrAsn                              1185119011951200                                                              LysIleAsnHisCysArgPheAspGluPhePheSerGluGlyCysAla                              120512101215                                                                  ProGlySerLysLysAspSerSerLeuCysLysLeuCysMetGlySer                              122012251230                                                                  GlyLeuAsnLeuCysGluProAsnAsnLysGluGlyTyrTyrGlyTyr                              123512401245                                                                  ThrGlyAlaPheArgCysLeuValGluLysGlyAspValAlaPheVal                              125012551260                                                                  LysHisGlnThrValProGlnAsnThrGlyGlyLysAsnProAspPro                              1265127012751280                                                              TrpAlaLysAsnLeuAsnGluLysAspTyrGluLeuLeuCysLeuAsp                              128512901295                                                                  GlyThrArgLysProValGluGluTyrAlaAsnCysHisLeuAlaArg                              130013051310                                                                  AlaProAsnHisAlaValValThrArgLysAspLysGluAlaCysVal                              131513201325                                                                  HisLysIleLeuArgGlnGlnGlnHisLeuPheGlySerAsnValThr                              133013351340                                                                  AspCysSerGlyAsnPheCysLeuPheArgSerGluThrLysAspLeu                              1345135013551360                                                              LeuPheArgAspAspThrValCysLeuAlaLysLeuHisAspArgAsn                              136513701375                                                                  ThrTyrGluLysTyrLeuGlyGluGluTyrValLysAlaValGlyAsn                              138013851390                                                                  LeuArgLysCysSerThrSerSerLeuLeuGluAlaCysThrPheArg                              139514001405                                                                  ArgPro                                                                        1410                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCTGTGGCCACCTGTCGCCCTGAC24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGCACACCATCTCACAGTTTTATCAGGGACCACAGCCTTGCAGGCCTTCGTGTGGGGGTC60                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCCTCGAAGCTGGTTCATCTG21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GACCCCCACACGAAGGCCTGCAAGGCTGTGGTCCTGATAAAACTGTGAGATGGTGTGCA59                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TGCACACCATCTCACAGTTTTATCAGGGACGACCTTTAGCCTGACGGT48                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCAGTGGCCCAATGGCATC19                                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAGGAGACATCCACCGTCAGGCTAAAGGTCGTCCCTGATAAAACTGTGAGA51                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CTTCCCATGAGGAGAGCT18                                                          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What is claimed is:
 1. A chimeric protein comprising a functional domainand a carrier domain, wherein the functional domain comprises an aminoacid sequence of a ligand-binding domain of a human low densitylipoprotein receptor which binds low density lipoprotein cholesterol andthe carrier domain comprises an amino acid sequence of human transferrinwhich binds a human transferrin receptor.
 2. DNA encoding a chimericprotein comprising a functional domain and a carrier domain, wherein thefunctional domain comprises an amino acid sequence of a ligand-bindingdomain of a human low density lipoprotein receptor which binds lowdensity lipoprotein cholesterol and the carrier domain comprises anamino acid sequence of human transferrin which binds a human transferrinreceptor.
 3. An expression plasmid comprising:a) a promoter; b) DNAencoding a chimeric protein comprising a functional domain and a carrierdomain, wherein the functional domain comprises an amino acid sequenceof a ligand-binding domain of a human low density lipoprotein receptorwhich binds low density lipoprotein cholesterol and the carrier domaincomprises an amino acid sequence of human transferrin which binds ahuman transferrin receptor,wherein expression of the DNA encoding thechimeric protein is under the control of the promoter.
 4. A geneticallymodified mammalian cell which expresses DNA encoding a chimeric protein,wherein the chimeric protein comprises a functional domain and a carrierdomain, wherein the functional domain comprises an amino acid sequenceof a ligand-binding domain of a human low density lipoprotein receptorwhich binds low density lipoprotein and the carrier domain comprises anamino acid sequence of human transferrin which binds a human transferrinreceptor.
 5. The genetically modified mammalian cell of claim 4, whereinthe mammalian cell is selected from the group consisting of: humancells, porcine cells, hamster cells, bovine cells, canine cells, mousecells, rat cells, monkey cells, feline cells, rabbit cells, sheep cellsand chimpanzee cells.
 6. The genetically modified human cell of claim 5,wherein the human cell is selected from the group consisting of: humanfibroblasts, human keratinocytes, human epithelial cells, human ovarycells, human endothelial cells, human glial cells, human neural cells,formed elements of the blood, human muscle cells, human hepatocytes andprecursors of these human cell types.
 7. The genetically modifiedmammalian cell of claim 5, wherein the mammalian cells are Chinesehamster ovary cells.
 8. The genetically modified mammalian cell of claim5, wherein the cell is an immortalized cell.
 9. The genetically modifiedhuman cell of claim 6, wherein the cell is a primary cell or a secondarycell.
 10. A pharmaceutical composition comprising the chimeric proteinof claim 1.